Mathilde Sarret

Pierre Adam
email: padam@unistra.fr

Philippe Schaeffer

Quentin Ebert

Julien Perthuison

Geneviève Pierrat-Bonnefois

BCE): Mode of preparation, alteration processes and botanical (re

Keywords: Ancient Egypt, Sesquiterpenoids, Cedar, Juniper, Oxidation, Archaeometry, Cedrium

published or not. The documents may come

Organic substances from Egyptian jars of the Early Dynastic period (3100-2700

Introduction

Archaeological excavations of the tombs of Kings from the two first Egyptian dynasties (3100-2700 B.C.) in Abydos (Egypt) in the late 19 th century directed by Emile Amélineau (Amelineau, 1901(Amelineau, , 1902(Amelineau, , 1904) ) led to the discovery of a series of ceramics. Several of the jars discovered are presently located in the Louvre Museum in Paris. They were postulated having been imported from the Levant based on the typology relating them to the so-called "Abydos ware" [START_REF] Hennessy | Spectrographic analysis of the foreign pottery from the royal tombs of Abydos and early Bronze Age pottery of Palestine[END_REF][START_REF] Serpico | A report on the analysis of the contents of a cache of jars from the tomb of Djer[END_REF][START_REF] Braun | South Levantine early Bronze Age chronological correlations with Egypt in light of the Narmer Serekhs from Tel Erani and Arad: New interpretations[END_REF]. Three of them, of similar typology, contain a material made of sand particles and black amorphous, resin-like, grains, likely of organic nature, of unknown origin and function. Although several hypotheses have been proposed concerning the content of this type of jars, postulated to correspond to oil, perfume or coniferous resin [START_REF] Serpico | A report on the analysis of the contents of a cache of jars from the tomb of Djer[END_REF], there is, to our knowledge, only one molecular study dedicated to the investigation of the organic content of potteries from the same period [START_REF] Serpico | A report on the analysis of the contents of a cache of jars from the tomb of Djer[END_REF]. The latter, discovered by [START_REF] Petrie | The royal tombs of the earliest dynasties, Part II. The Egypt Exploration Fund[END_REF] in the tomb of Djer at Abydos, correspond to ceramics now being part of the collections of the British Museum, of the Petrie Museum of London, and of the Ashmolean Museum. The present molecular study aims at identifying the molecular composition of the resin-like material from the jars from Abydos using gas-chromatography-mass spectrometry (GC-MS), in order to assess its biological and geographical origin, as well as its use and mode of preparation. In addition, laboratory experiments involving essential oils from conifers were conducted in order to reproduce the oxidative alteration processes affecting sesquiterpenoids when exposed to air atmosphere upon ageing.

Experimental

Samples

The samples were collected in three jars found in the tombs of Kings from the two first Egyptian dynasties (3100-2700 B.C.) at Abydos (Egypt) by Emile Amélineau in 1895-1896.

The typology of the jars (Fig. 1) relates them to the so-called "Abydos ware" which comprises pottery vessels, jugs, and jars initially found in Abydos but later also in other places in Egypt [START_REF] Hennessy | Spectrographic analysis of the foreign pottery from the royal tombs of Abydos and early Bronze Age pottery of Palestine[END_REF]. The jars are presently located in the Musée du Louvre in Paris with the reference numbers E21727, E21809 and E28010.

In the case of jar E21727, sampling has been performed on the mixture of sand and organic material sticking on the neck of the jar. For the other two jars (E21809, E28010), the samples were collected by pouring the jars content on an aluminum foil. The samples obtained had a similar aspect and consisted of sand particles mixed with black amorphous grains, the latter being almost completely soluble in organic solvents.

Extraction and fractionation

Two distinct extraction protocols have been developed for the analysis of the resin-like material, the first one (protocol A, see below) involving derivatization which allows investigation of polar lipids like alcohols and carboxylic acids, but results in the loss of the more volatile compounds from the extract, and the second one (protocol B), devoid of derivatization steps to avoid evaporation steps, giving access to volatile sesquiterpenoids, but not to the more polar compounds.

Extraction using protocol A

The samples (70 mg, 80 mg, 606 mg, for E21727, E21809 and E28010 respectively) were extracted by sonication with a mixture of CH 2 Cl 2 /CH 3 OH (1:1, v/v). An aliquot of each crude extract obtained by filtration on celite was successively acetylated (Ac 2 O/Pyridine 1:1 v/v, 60°C, 2 h) and, after removal of the solvent and excess reagent under reduced pressure, esterified using a solution of diazomethane in Et 2 O. The derivatized extracts were fractionated by liquid chromatography (LC) on silica gel eluting successively with a mixture of CH 2 Cl 2 /EtOAc (8:2 v/v) and CH 2 Cl 2 /CH 3 OH (6:4 v/v), leading, respectively, to a moderately polar fraction F1 analysed using GC-flame ionization detection (GC-FID) and GC-MS, and a polar fraction F2 not being investigated further.

Extraction using protocol B

The samples (573 mg, 587 mg, 426 mg, for E21727, E21809 and E28010 respectively) were extracted by sonication with CH 2 Cl 2 . The crude extracts obtained by filtration on celite were concentrated under reduced pressure, avoiding complete solvent removal to preserve the volatile compounds. The extract was chromatographed over silica gel using CH 2 Cl 2 as eluent in order to recover an apolar fraction (F1 volatile ) and remove the polar constituents of the extract, and F1 volatile was concentrated under reduced pressure and analysed by GC-FID and GC-MS.

Identification of key components

Several aromatic (poly)carboxylic acids and sesquiterpenic structures have been identified using reference compounds. Details on the preparation and characterisation of these standards are described in detail in the Supplementary data. were heated at 70 °C under an atmosphere of O 2 in a glass tube (10 ml) closed with a screwcap.

Oxidation of essential oil of Cedrus atlantica

After 20 h, H 2 O (400 l) was added to an aliquot (500 l) of the reaction mixture. The organic compounds were extracted with pentane (400 l) and analyzed by GC-MS without further purification.

Analytical methods

GC-FID

GC-FID was carried out with a Hewlett Packard 6890 gas chromatograph equipped with an oncolumn injector, FID and a HP-5 fused silica capillary column (30 m x 0.32 mm; 0.25 m film thickness). H 2 was used as carrier gas (constant flow mode, 2.5 ml/min), and the oven was programmed as follows: Temperature program: 40 °C (10 min -1 ), 40 °C-300 °C (5 °C min -1 ), isothermal at 300 °C for 30 min.

GC-MS

GC-MS was carried out with a Thermo Scientific Trace Ultra gas chromatograph coupled to a Thermo Scientific TSQ Quantum mass spectrometer equipped with an autosampler Tri Plus and a programmed temperature vaporizing (PTV) injector. The temperature of the source was set at 220 °C. The mass spectrometer was operating in the electron impact (EI) mode at 70 eV and scanning m/z 50 to 700. Gas chromatographic separations were performed on a HP5-MS column (30 m x 0.25 mm; 0.1 m film thickness) using He as carrier gas. Temperature program:

40 °C (10 min -1 ), 40 °C-300 °C (5 °C min -1 ), isothermal at 300 °C for 30 min. The data were investigated using the Xcalibur Software.

Nuclear Magnetic Resonance (NMR)

The NMR spectra were recorded on a Bruker Avance I 500 MHz spectrometer (500 MHz for 1 H; 125 MHz for 13 C). The chemical shifts are reported in ppm relative to tetramethylsilane with the solvent used as internal standard (CHDCl 2 :  1 H 5.32 ppm; CD 2 Cl 2 :  13 C 53.8 ppm)

and the coupling constants are expressed in Hz.

Results and discussion

GC-MS investigation of the lipid extracts

The distributions of the apolar fraction (F1) from the three samples obtained using protocol A (cf. § 2.3) revealed the presence of a highly complex assemblage of compounds (Fig. 2a for sample E21727, and Fig. S1 in Supplementary data for samples E21809 and 28010). Four main series of compounds could be detected comprising terpenic structures, aromatic carboxylic acids, fatty acids and phenolic compounds. Since most of these compounds had carbon skeleton ≤ 15 C atoms, a second protocol (protocol B) optimized to avoid the loss of the more volatile constituents has been used and the recovered apolar fractions (F1 volatile ) were analyzed by GC-MS. Molecular distributions of the F1 volatile fractions were again very similar to each other with terpenic structures predominating (Fig. 2b and Fig. S2 in Supplementary data). Several compounds from fractions F1 and F1 volatile could be firmly identified by comparison of mass spectra and GC behavior with those of commercial standards or compounds obtained by preparation of references (cf. Supplementary data).

Sesquiterpenic structures

The distributions obtained (Fig. 2a,b) are dominated by four main families of sesquiterpenoid derivatives: (a) tricyclic sesquiterpenoids comprising, notably, longiborneol 1 (numbers refer to structures in the Appendix), its related ketone (longicamphor) 2, and longicamphenilone 3 related to longifolene 4 (cf. Fig S .3 and Section 2.1 from Supplementary data). (b) sesquiterpenoids from the cuparene series (cf. Fig. S4d and Section 2.2. from Supplementary data) which include cuparene 5, cuparenic acid 6 [START_REF] Enzell | The chemistry of the natural order Cupressales -XXI. Cuparene and cuparenic acid, two sesquiterpenic compounds with a new carbon skeleton[END_REF] identified by comparison with its mass spectrum published by Grantham and Douglas (1980) and a ketone 7 (Fig. S4c in Supplementary data), the mass spectrum of which being identical to that of -cuparenone 7a published in the NIST library. Since it is not possible to infer the exact position of the carbonyl group on the cyclopentane ring using MS, this compound might also correspond to -cuparenone 7b. A further compound having a mass spectrum very similar to that of cuparenic acid 6 (presence of an even fragment at m/z 176 for the related methyl ester) but with a molecular ion shifted upwards by 14 Da (Fig. S4d in Supplementary data) could correspond to the analogue 8 of cuparenic acid 6 bearing a carbonyl group on the cyclopentane ring at a position which could not be determined but possibly similar to that of compound 7a or 7b. (c) cadalene 9 which might be formed from the various sesquiterpenoids of the cadinene series (e.g. 10, 11) by an aromatization process.

(d) a series of aromatic and oxygenated sesquiterpenoids (12-15) related to himachalenes (16)(17)(18). Sesquiterpenoids 12-15 were unambiguously identified by comparison of mass spectra (Fig. 3) and GC behavior with those of reference compounds prepared by oxidation of himachalene-related sesquiterpenoids from the essential oil of Cedrus atlantica (Fig S .5 and Section 2.3 from Supplementary data). Firm determination of the structure of these compounds using NMR spectroscopy of isolated compounds constitutes the first unambiguous identification of himachalene-related biomarkers in archaeological samples.

Diterpenoids

Diterpenoids were detected in low relative amounts (Fig. 2a,b), and comprised mainly 7 oxodehydroabietic acid 19 and 7-oxo-dehydroabietane 20 identified on the basis of their mass spectra [START_REF] Van Den Berg | Analysis of diterpenoid resins and polymers in paint media and varnishes with an atlas of mass spectra[END_REF][START_REF] Bhar | Novel domino reactions for diterpene synthesis[END_REF]. A phenolic diterpenoid, totarol 21, identified using an authentic standard, was also present in one sample (E21809; Fig S2 in Supplementary data).

Benzene polycarboxylic acids and acetophenone derivatives

Benzene(poly)carboxylic acids were predominant in the organic extracts of samples E21727 and E21809, but were relatively less abundant in sample E28010 (Fig. 2a; Fig. S1 in Supplementary data). Compounds 22-26 were unambiguously identified using prepared standards (cf. Section 2.4 from Supplementary data), allowing the precise position of the substituents on the aromatic ring to be determined. Their formation can be explained by autoxidation at the oxygen-sensitive benzylic positions (e.g. [START_REF] Mayo | Free-radical autoxidation of hydrocarbons[END_REF][START_REF] Charrié-Duhaut | Abiotic oxidation of bitumen under natural conditions[END_REF] from aromatic terpenic structures deriving from biological unsaturated sesquiterpenes and/or monoterpenoids as illustrated in Fig. 4 (cf. Section 3.2 and Fig. 5 and 6 below dedicated to oxidation processes of terpenoids). The occurrence of compounds 25-26 has been reported

previously from the organic residue of a broken jar found in a tomb of the XXVI th Dynasty by [START_REF] Seher Von | Untersuchung von "Ölproben" aus archäologischen Funden[END_REF], these compounds being interpreted as oxidation products of terpenoids. p-Methylacetophenone 27 detected in the volatile fraction (Fig. 2b) can similarly be the result of the oxidation of monocyclic mono-and sesquiterpenoids. Vanillic acid 28 has likely another origin which will be discussed in Section 3.4.

Fatty acids

Fatty acids (FA) were minor constituents of the samples (cf Fig. 2a; Fig. S1). Their distribution was dominated by palmitic (C 16 ) and stearic (C 18 ) acid. Longer chain FA (C 22 -C 28 )

indicating a contribution from plants appear in trace amounts. No typical oxidation products of unsaturated FA such as carboxylic acid diols or diacids [START_REF] Colombini | Characterisation of organic residues in pottery vessels of the Roman age from Antinoe (Egypt)[END_REF][START_REF] Hansel | Formation of dihydroxy acids from Z-monounsaturated alkenoic acids and their use as biomarkers for the processing of marine commodities in archaeological pottery vessels[END_REF] were detected. The relatively low proportion of these compounds as compared to the sesquiterpenic structures and to the aromatic (poly)carboxylic acids suggests that oil or fat are not part of the ingredients used to prepare the substances from the jars.

Oxidation processes of terpenoids

The predominance of oxidized analogues of sesquiterpenoids from the himachalene and cuparene series, together with the occurrence of longicamphenylone 3 and longicamphor 2possible oxidation products of longifolene 4 and longiborneol 1, respectively -(Fig. 5a), as well as the predominance of benzene polycarboxylic acids and acetophenone derivatives ( § 3.1), attest of the intense oxidative alteration processes to which the organic substances in the jars were submitted to since their preparation. The intensity of such an alteration is further underlined by the absence of sesquiterpenoids with non-conjugated double bonds, which generally predominate among genuine plant terpenoids, but are particularly sensitive to oxidative alteration (e.g. [START_REF] Ngo | Autoxidation of -santalene[END_REF][START_REF] Huang | Mechanistic studies on the autoxidation of -guaiene: Structural diversity of the sesquiterpenoid downstream products[END_REF]. This is the case of sesquiterpenoids of the himachalene series, naturally-occurring as himachalene dienes (16-18) which could not be detected. All the himachalene derivatives found in the extracts (12-15) most likely corresponded to products formed by alteration processes (Fig. 5b), although arhimachalene 12 sometimes occurs in small amounts in plant extracts (e.g. [START_REF] Bredenberg | Sesquiterpenes from Cedrus species[END_REF]Panday and Dev, 1968;[START_REF] Baser | The essential oil of Taurus cedar (Cedrus libani A. Rich): Recent results[END_REF][START_REF] Loizzo | Composition and -amylase inhibitory effect of essential oil from Cedrus libani[END_REF]. It thus appears that the original sesquiterpenic distributions of the investigated organic substances have been severely altered, most of the sesquiterpenoids initially present have completely disappeared or have been converted into oxidized analogues. The origin of the oxidized cuparenes 6-8 can only partly be ascribed to autoxidation processes (Fig. 5c). If cuparenic acid 6 likely results from the autoxidation at the benzylic methyl group of cuparene 5, abiotic oxidative processes are, however, not expected to be involved in the formation of cuparenone 7 since such processes would involve autoxidation at a non-activated methylenic position. Therefore, it has to be envisaged that cuparenone 7 is a naturally-occurring compound directly originating from a vegetal source used to prepare the organic substances from the jars. and -cuparenones 7a and 7b have been reported only from tree species not native from the Eastern Mediterranean region (e.g. Thuija orientalis; [START_REF] Dev | Ketones from "Mayur Pankhi": Some new cuparene-based terpenoids[END_REF] and which were probably not available in ancient Egypt and in the Levant.

Alternatively, it can be proposed that incorporation of the keto group on the cyclopentane ring of cuparene 5 was induced by biotic oxidation. In this respect, the biotransformation of cuparene by Aspergillus niger has been shown to result in the incorporation of oxygenated functions (alcohols, carbonyls) on the cyclopentane ring of cuparene [START_REF] Asakawa | Biotransformation of essential oils, ionones, damascones, adamantanes, aromatic compounds by green algae, fungi and mammals[END_REF] and in the oxidation of the aromatic methyl group into a carboxylic acid, thus providing an alternative biotic (as opposed to abiotic) formation pathway for both cuparenic acid 6, compound 8 and possibly compound 7.

In order to evaluate in greater detail the impact of abiotic oxidation processes on the distribution of sesquiterpenoids from the himachalene series, a laboratory experiment involving the essential oil of C. atlantica, a radical initiator (NHPI and Co(acac) 2 ) under an oxygen atmosphere [START_REF] Ishii | Alkane oxidation with molecular oxygen using a new efficient catalytic system: N-hydroxyphthalimide (NHPI) combined with Co(acac) n (n=2 or 3)[END_REF][START_REF] Matsushita | Aerobic oxidation of 8,11,13-abietatrienes catalyzed by N-hydroxyphthalimide combined with 2,2azobis(4-methoxy-2,4-dimethylvaleronitrile) and its application to synthesis of naturally occurring diterpenes[END_REF] has been performed to simulate abiotic autoxidation processes. Under these conditions, himachalenes 16-18, the predominant sesquiterpenoids from the original essential oil (Fig. 6a), have completely disappeared after 20 h of reaction at 70 °C (Fig. 6b). Concomitantly, the relative abundance of several compounds, initially present in trace amounts, comprising ar-himachalene 12 and its unsaturated analogues (29, 30), increased significantly (Fig. 6b). Given their low initial concentration, they most likely formed by dehydrogenation processes, involving, for instance, the successive incorporation of oxygen followed by elimination of water or hydrogen peroxide. In addition, it was also possible to observe the formation of oxygenated derivatives of ar-himachalene, 13-15 (Fig. 5b), which were also detected among the terpenoid derivatives from the three jars (Fig. 2b). These compounds can thus be interpreted as oxidation products of the unsaturated analogues of arhimachalene 29 and 30 (Fig. 5b).

Botanical and geographical origin of the substances from the jars

Several lipid constituents from the organic extracts of the jars could be unambiguously related to terpenoids from conifers. Such is the case of longiborneol 1, longicamphor 2, longicamphenylone 3 (Fig. 2b) most likely formed by oxidation of longifolene 4 -a non-specific sesquiterpene from conifers -, cadalene 9 -an aromatized analogue of cadinene-type terpenoids widely distributed in conifers [START_REF] Otto | Sesqui-,di-, and triterpenoids as chemosystematic markers in extant conifers[END_REF]) -, as well as 7 oxo-dehydroabietic acid 19 and of 7-oxo-dehydroabietatriene 20 -oxidized analogues of abietic acid 31 and of abietadiene 32 or abietatriene 33 abundant in conifers (e.g.. [START_REF] Otto | Terpenoids as chemosystematic markers in selected fossil and extant species of pine (Pinus, Pinaceae)[END_REF][START_REF] Otto | Sesqui-,di-, and triterpenoids as chemosystematic markers in extant conifers[END_REF]. However, these compounds are not specific and cannot be related to any precise conifer species or genus. The very low amounts of 7-oxo-dehydroabietic acid 19 detected in the samples clearly indicate that conifer resins, generally dominated by diterpenic acids from the abietane (34) and pimarane ( 35) series (e.g., [START_REF] Keeling | Diterpene resin acids in conifers[END_REF], can be ruled out as ingredients. Himachalene (12-15) and cuparene (5-8) derivatives, as well as totarol 21, are more specific and allow some conclusions to be drawn regarding the nature of the conifers used to prepare the substances from the jars. Thus, if we consider potential conifer sources from the Eastern Mediterranean region [START_REF] Berjaoui | La distribution des essences forestières au Liban[END_REF][START_REF] Germer | Flora des pharaonischen Ägypten[END_REF][START_REF] Quézel | Diversité et répartition des sapins sur le pourtour méditerranéen[END_REF][START_REF] Serpico | Resin, amber and bitumen[END_REF][START_REF] Hajar | Cedrus libani (A. Rich) distribution in Lebanon: Past, present and future[END_REF]Caudullo and de Rigo, 2016a,b,c,d), himachalenes are biosynthesized in particularly high amounts by Cedrus libani (e.g. [START_REF] Agrawal | Chemistry of true cedars[END_REF][START_REF] Fleisher | The volatiles of the leaves and wood of Lebanon cedar (Cedrus libani A. Rich) aromatic plants of the Holy Land and the Sinai[END_REF][START_REF] Saab | Essential oil components in heart wood of Cedrus libani and Cedrus atlantica from Lebanon[END_REF] and, to a more limited extent, by Abies cilicica [START_REF] Bagci | Study of the essential oil composition of two subspecies of Abies cilicica (Ant. et Kotschy) Carr. from Turkey[END_REF]. Additionally, members of the Cupressaceae family also produce himachalenes, notably several juniper species (e.g., Juniperus communis: [START_REF] Gonny | Analysis of Juniperus communis subsp. Alpine needle, berry, wood and root oils by combination of GC-GC-MS and 13 C-NMR[END_REF]J. phoenicea: Chalchat et al., 1988;J. oxycedrus: Saab et al., 2012) but not cypresses such as the Mediterranean cypress (Cupressus sempervirens). Cuparene derivatives are typical sesquiterpenes from Cupressaceae such as cypresses [START_REF] Piovetti | Volatile constituents of Cupressus dupreziana and the sesquiterpenes of Cupressus sempervirens[END_REF][START_REF] Otto | Sesqui-,di-, and triterpenoids as chemosystematic markers in extant conifers[END_REF] and junipers [START_REF] Feliciano | Structure elucidation of germacrane alcohols from Juniperus communis subsp. hemisphaerica[END_REF][START_REF] Otto | Sesqui-,di-, and triterpenoids as chemosystematic markers in extant conifers[END_REF][START_REF] Seca | The chemical composition of Juniperus genus (1970-2004)[END_REF][START_REF] Gonny | Analysis of Juniperus communis subsp. Alpine needle, berry, wood and root oils by combination of GC-GC-MS and 13 C-NMR[END_REF][START_REF] Robbat | Tracking juniper berry content in oils and distillates by spectral deconvolution of gas chromatography/mass spectrometry data[END_REF][START_REF] Saab | Phytochemical analysis and cytotoxicity towards multidrug resistant leukemia cells of essential oils derived from Lebanese medicinal plants[END_REF], and cuparene 5 is biosynthesized in significant proportions relative to other sesquiterpenoids by both Juniperus sp. and Cupressus sp. from the Eastern Mediterranean region comprising J. oxycedrus (Chalchat et al., 1988, Uçar and[START_REF] Uçar | The composition of volatile extractives from the wood of Juniperus excelsa, Juniperus foetidissima and Juniperus oxycedrus[END_REF], J. phoenicea [START_REF] Chalchat | Chemical compositions of the hexane fraction of empyreumatic wood oils from Juniperus oxycedrus L. and Juniperus pheonicea L[END_REF], J. drupacea [START_REF] Adams | Comparisons of the leaf oils of Juniperus drupacea Labill. from Greece, Turkey and the Crimea[END_REF], J. excelsa [START_REF] Uçar | The composition of volatile extractives from the wood of Juniperus excelsa, Juniperus foetidissima and Juniperus oxycedrus[END_REF][START_REF] Saab | Phytochemical analysis and cytotoxicity towards multidrug resistant leukemia cells of essential oils derived from Lebanese medicinal plants[END_REF], J. foetidissima [START_REF] Uçar | The composition of volatile extractives from the wood of Juniperus excelsa, Juniperus foetidissima and Juniperus oxycedrus[END_REF], J.

communis [START_REF] Gonny | Analysis of Juniperus communis subsp. Alpine needle, berry, wood and root oils by combination of GC-GC-MS and 13 C-NMR[END_REF], and C. sempervirens [START_REF] Piovetti | Volatile constituents of Cupressus dupreziana and the sesquiterpenes of Cupressus sempervirens[END_REF]. To our knowledge, except in two studies reporting the presence of cuparene 5 in trace amounts in C. libani [START_REF] Baser | The essential oil of Taurus cedar (Cedrus libani A. Rich): Recent results[END_REF] and C. atlantica [START_REF] Aberchane | Analysis of Moroccan Atlas cedarwood oil (Cedrus atlantica Manetti)[END_REF], cuparene derivatives are not reported from real cedars (e.g., [START_REF] Agrawal | Chemistry of true cedars[END_REF][START_REF] Fleisher | The volatiles of the leaves and wood of Lebanon cedar (Cedrus libani A. Rich) aromatic plants of the Holy Land and the Sinai[END_REF][START_REF] Otto | Sesqui-,di-, and triterpenoids as chemosystematic markers in extant conifers[END_REF][START_REF] Loizzo | Composition and -amylase inhibitory effect of essential oil from Cedrus libani[END_REF][START_REF] Paoli | Chemical variability of wood essential oil of Cedrus atlantica Manetti from Corsica[END_REF][START_REF] Saab | Phytochemical analysis and cytotoxicity towards multidrug resistant leukemia cells of essential oils derived from Lebanese medicinal plants[END_REF].

The predominance of aromatized and oxidized compounds such as 12-15 deriving from In the latter case, one may envisage that the structure of cuparene, which does not contain nonconjugated double bonds and has a limited number of oxygen-sensitive non-quaternary benzylic positions in contrast to himachalene derivatives, would confer to this compound an enhanced relative resistance upon oxidative ageing. However, this enhanced resistance is unlikely to be sufficient for resulting in such a clear predominance of cuparene-related compounds in the altered material which only contained trace amounts (if any) of cuparene within the genuine products. Therefore, we propose that cuparene-related compounds originate from vegetal sources other than real cedars, such as junipers or cypresses (Cupressaceae) which biosynthesize cuparene 5 in significant high relative amounts. This possibility is further supported by the detection of totarol 21 in one sample (E21809, Fig. S1 in Supplementary data), this phenolic compound being present in Juniperus sp. and Cupressus sp. (e.g. [START_REF] Feliciano | Diterpenoides neutros y compuestos aromaticos de las hojas de Juniperus phoenicea subsp[END_REF][START_REF] Gonny | Analysis of Juniperus communis subsp. Alpine needle, berry, wood and root oils by combination of GC-GC-MS and 13 C-NMR[END_REF][START_REF] Dob | Chemical composition of the essential oil of Juniperus phoenicea L. from Algeria[END_REF][START_REF] Herzi | Extraction of essential oil from Cupressus sempervirens: comparison of global yields, chemical composition and antioxidant activity obtained by hydrodistillation and supercritical extraction[END_REF]). An origin of totarol 21 from real cedars might however be considered given the report of this compound in the leaves of C.

atlantica (Lercker et al., 1977, Agrawal and[START_REF] Agrawal | Chemistry of true cedars[END_REF], although our own investigations could not confirm this finding, neither could we detect totarol in the leaves and twigs of C.

libani. In addition, this compound has never been reported from C. libani wood.

To conclude, we propose that the organic substances from the jars from the Early Dynastic period predominantly originate from C. libani with a contribution from a member of the Cupressaceae family (Cupressus sp or Juniperus sp.) to account for the presence of the various cuparene derivatives and of totarol. Except J. phoenicea which grows in the Sinai [START_REF] Germer | Flora des pharaonischen Ägypten[END_REF]Serpico andWhite, 2000, Caudullo, G. andde Rigo, 2016a.), the various Cedrus, Juniper and Abies species mentioned above do not grow in Egypt [START_REF] Berjaoui | La distribution des essences forestières au Liban[END_REF][START_REF] Germer | Flora des pharaonischen Ägypten[END_REF][START_REF] Quézel | Diversité et répartition des sapins sur le pourtour méditerranéen[END_REF][START_REF] Serpico | Resin, amber and bitumen[END_REF][START_REF] Hajar | Cedrus libani (A. Rich) distribution in Lebanon: Past, present and future[END_REF]Caudullo and de Rigo, 2016a,b,c,d),

but are all native from the Levant. Therefore, the organic material from the jars has likely been prepared in southern Levant, where the jars are postulated to originate from (Hennessy and Millet, 1963;[START_REF] Serpico | A report on the analysis of the contents of a cache of jars from the tomb of Djer[END_REF][START_REF] Braun | South Levantine early Bronze Age chronological correlations with Egypt in light of the Narmer Serekhs from Tel Erani and Arad: New interpretations[END_REF].

Implications on the constitution of "cedrium"

The organic substances from the jars present many molecular similarities with the material referred to as "oil of cedar" or "cedrium" in the literature and used as an ingredient for embalming [START_REF] Buckley | Complex organic chemical balms of pharaonic animal mummies[END_REF][START_REF] Koller | Analysis of a pharaonic embalming tar[END_REF][START_REF] Koller | Herodotus' and Pliny's embalming materials identified on ancient Egyptian mummies[END_REF]Koller et al., , Łucejko et al., 2012;;[START_REF] Scholz-Böttcher | An 18th century "Mumia vera aegyptica" -Fake or authentic?[END_REF]Brettel et al., 2016). In particular, the present organic material and "cedrium" are both characterized by the significant contribution of cuparene derivatives as reported in the latter case by several autors (cf. Koller et al.;[START_REF] Van Den Berg | Analysis of diterpenoid resins and polymers in paint media and varnishes with an atlas of mass spectra[END_REF], 2005;[START_REF] Łucejko | An analytical approach based on X-ray diffraction Fourier transform infrared spectroscopy and gas chromatography/mass spectrometry to characterize Egyptian embalming material[END_REF][START_REF] Scholz-Böttcher | An 18th century "Mumia vera aegyptica" -Fake or authentic?[END_REF]Brettel et al., 2016). Additionally, the "cedrium" described by [START_REF] Koller | Herodotus' and Pliny's embalming materials identified on ancient Egyptian mummies[END_REF] 2013). Thus, we propose, based on the comparison of the mass spectra of the himachalene derivatives identified in our samples (cf Fig. 3) with mass fragmentation patterns reported in Figure 2 and 4 in [START_REF] Koller | Herodotus' and Pliny's embalming materials identified on ancient Egyptian mummies[END_REF] and Table 4 in Scholz-Böttcher et al.

(2013), that "cadinatriene" most likely corresponds to ar-himachalene 12, "anhydro-a-copaene" to ketone 13, "tetramethoxyphenol" to ar-himachalene-related unsaturated ketone 15, and "2acetyl-6-methoxynaphtalene" to dehydro-ar-himachalene 29 or 30. Identical mass spectra were also reported by [START_REF] Hamm | Headspace solid phase microextraction for screening for the presence of resins in Egyptian archaeological samples[END_REF] to occur for lipids recovered using headspace solid phase extraction of organic material from the embalming of a heart found in a blue vase from the Louvre Museum (reference numbers E 11094, cf. [START_REF] Charrié-Duhaut | The canopic jars of Ramesses II : real use revealed by molecular study of organic residues[END_REF]. "Cedrium" is thus, from a molecular point of view, very similar to the samples from the jars investigated, both being characterized by predominant sesquiterpenoids from the cuparene and himachalene series originating from a plant mixture comprising Cedrus sp. and a member of the Cupressaceae family.

Our results and comparison with those obtained from previous molecular studies on embalming material from more recent periods give clues to a controversy discussed by [START_REF] Buckley | Complex organic chemical balms of pharaonic animal mummies[END_REF] and [START_REF] Koller | Analysis of a pharaonic embalming tar[END_REF][START_REF] Koller | Herodotus' and Pliny's embalming materials identified on ancient Egyptian mummies[END_REF] concerning the use of cedar oil for embalming mummies. This controversy begun when [START_REF] Lucas | Cedar"-tree products employed in mummification[END_REF] pointed out that the so called "cedar oil" corresponds to a material extracted from juniper. [START_REF] Wisseman | Preserved for the afterlife[END_REF] also mentioned that juniper oil was probably used as an ingredient for mummification rather than cedar oil. This hypothesis, which also appeared in more recent articles (e.g. Abdel-Maksoud and El-Amin; 2011) does, however, not rely on detailed molecular investigation or on the detection of juniper-specific molecular markers. [START_REF] Wisseman | Preserved for the afterlife[END_REF] did not exclude that further investigations of embalming material might show that the use of cedar vs. juniper oil may have changed with time, depending on criteria such as fashion or cost. Other authors, on the other hand, favored the hypothesis of the use of cedar oil originating from Cedrus sp based on molecular criteria, although the latter have proven to be ambiguous and tenuous. Thus, based on MS interpretations, [START_REF] Buckley | Complex organic chemical balms of pharaonic animal mummies[END_REF] have identified two oxidized derivatives of himachalenes, the predominant sesquiterpenes of oils from Cedrus sp. (e.g. [START_REF] Agrawal | Chemistry of true cedars[END_REF][START_REF] Fleisher | The volatiles of the leaves and wood of Lebanon cedar (Cedrus libani A. Rich) aromatic plants of the Holy Land and the Sinai[END_REF][START_REF] Otto | Sesqui-,di-, and triterpenoids as chemosystematic markers in extant conifers[END_REF][START_REF] Loizzo | Composition and -amylase inhibitory effect of essential oil from Cedrus libani[END_REF][START_REF] Paoli | Chemical variability of wood essential oil of Cedrus atlantica Manetti from Corsica[END_REF][START_REF] Saab | Phytochemical analysis and cytotoxicity towards multidrug resistant leukemia cells of essential oils derived from Lebanese medicinal plants[END_REF] in the material used to embalm a cat mummy, but the authors recognized that the identification was only tentative and that they could unfortunately not firmly support the hypothesis that the embalming material indeed contained true cedar oil. [START_REF] Koller | Analysis of a pharaonic embalming tar[END_REF][START_REF] Koller | Herodotus' and Pliny's embalming materials identified on ancient Egyptian mummies[END_REF]) also discussed the use of cedar vs. juniper oil for mummification. These authors proposed a contribution from cedar and excluded the use of juniper given the absence of cedrol 36 among the sesquiterpenoids from an unused embalming material unearthed at Deir el-Bahari (ca. 1500 BC), whereas this compound is reported to occur in fresh juniper extracts (e.g. [START_REF] Piovetti | Volatile constituents of Cupressus dupreziana and the sesquiterpenes of Cupressus sempervirens[END_REF][START_REF] Uçar | The composition of volatile extractives from the wood of Juniperus excelsa, Juniperus foetidissima and Juniperus oxycedrus[END_REF]. However, cedrol is not necessarily abundant and can even be absent from junipers (e.g. [START_REF] Uçar | The composition of volatile extractives from the wood of Juniperus excelsa, Juniperus foetidissima and Juniperus oxycedrus[END_REF][START_REF] Gonny | Analysis of Juniperus communis subsp. Alpine needle, berry, wood and root oils by combination of GC-GC-MS and 13 C-NMR[END_REF]. The contribution of cedar was established from the identification by MS of cuparene 5, cadalene 9, cadinatriene or acetylmethoxynaphthalene. The argument raised by the authors was, however, not completely convincing for the following reasons: (1) cuparene 5 and related compounds most likely originate from Cupressaceae (e.g. Otto and Wilde and references cited therein) rather than from real cedar. As discussed above, the relative low proportions of cuparene 5 in essential oils of C.

libani (a single report, by [START_REF] Baser | The essential oil of Taurus cedar (Cedrus libani A. Rich): Recent results[END_REF] can probably not explain the importance of these compounds in our extracts, unless an extreme and unrealistic enrichment relative to other sesquiterpenoid series due to an enhanced resistance of cuparene 5 towards oxidative alteration is envisaged; (2) cadalene 9 and cadinatriene may originate from the aromatization/dehydrogenation of sesquiterpenoids from the cadinene series (e.g. 10, 11), the latter being non-specific of real cedars and being also found in junipers [START_REF] Seca | The chemical composition of Juniperus genus (1970-2004)[END_REF];

(3) acetylmethoxynaphthalene has not been reported from real cedars. However, it appears following the present identification work -cf. discussion above -that some of these structures were erroneously attributed by the authors with cadinatriene and acetylmethoxynaphthalene, for instance, corresponding in fact to the himachalene derivatives (12, 29 or 30) identified in the present study, which indeed supports the hypothesis of a significant contribution of material originating from C. libani.

To conclude, comparison of the results of the present molecular study with previous studies on embalming material from more recent historical periods leads to the conclusion that the material called "cedrium" used for embalming originates from true cedar (most likely C. libani) mixed with a second botanical source belonging to the Cupressaceae family, possibly cypresses and/or junipers.

Use of the organic material from the jars

Given the molecular analogies of the organic substances in the jars with "cedrium", one might envisage that they were used as an ingredient for embalming as reported by several authors [START_REF] Buckley | Complex organic chemical balms of pharaonic animal mummies[END_REF][START_REF] Koller | Analysis of a pharaonic embalming tar[END_REF][START_REF] Koller | Herodotus' and Pliny's embalming materials identified on ancient Egyptian mummies[END_REF]Koller et al., , Łucejko et al., 2012;;[START_REF] Scholz-Böttcher | An 18th century "Mumia vera aegyptica" -Fake or authentic?[END_REF]Brettel et al., 2015). However, true embalming only began in Egypt after 2500 BC, although recent work indicates some earlier examples of mummification, before 2900 BC [START_REF] Jones | Evidence for prehistoric origins of Egyptian mummification in late Neolithic burials[END_REF]. Therefore, the use of the odoriferous substances from the jars dating to the Early Dynastic period for embalming remains rather uncertain, and alternate uses, for cosmetology or medical and cultual purposes have to be considered.

Mode of preparation of the substances identified in the jars

The predominance of low molecular constituents (e.g. sesquiterpenoids) suggests that the preparation of the organic material from vegetal sources most likely involves a concentration step of volatile compounds, which could be achieved by distillation. Consequently, one might first envisage that the substances from the jars correspond to wood tar, similar to cade oil, made by dry distillation. Although unambiguous markers for wood tar such as methyl, ethyl or propyl guaiacols 37-39 formed by pyrolysis of wood lignin (e.g. [START_REF] Faix | Thermal degradation products of wood -Gas chromatography separation and mass spectrometry characterization of monomeric lignin derived products[END_REF][START_REF] Bailly | Identification of alkyl guaiacyl dehydroabietates as novel markers of wood tar from Pinaceae in archaeological samples[END_REF] were not detected, it cannot be completely excluded that vanillic acid 28 identified in the samples (Fig. 2a and Fig. S1 in Supplementary data) corresponds to an oxidation product of alkylguaiacols 37-39 (Fig. 7) given the severe oxidative alteration undergone by the substances.

Alternatively, vanillic acid may result from the oxidation of low molecular weight methoxyphenols such as, e.g., vanillin 40 or eugenol 41 (Fig. 7) from the plants used as ingredients. In addition, the aromatic diterpenoids frequently encountered in conifer wood tar such as 18-and 19-nor abietatrienes 42, 43, and retene 44 related to abietic acid 31 and resulting from the aromatization of resin acids (e.g. [START_REF] Evershed | Pine wood origin for pitch from the Mary Rose[END_REF][START_REF] Beck | Ancient pine pitch: technological perspectives from a Hellenistic shipwreck[END_REF][START_REF] Egenberg | Organic geochemical evidence for pine tar production in Middle Eastern Sweden during the Roman Iron Age[END_REF][START_REF] Connan | Conifer tar on the keel and hull planking of the Ma'agan Mikhael Ship (Israel, 5th century BC): identification and comparison with natural products and artefacts employed in boat construction[END_REF][START_REF] Hjulström | Organic geochemical evidence for pine tar production in Middle Eastern Sweden during the Roman Iron Age[END_REF][START_REF] Connan | Etude géochimique d'enduits de surface, de tissus de calfatage, de blocs de poix et de bitume des épaves gallo-romaines du Parc Saint-Georges à Lyon[END_REF] could not be detected in the samples. Therefore, we propose that the substances from the jars correspond to essential oils obtained by steam distillation of plant material comprising wood, twigs, cones or berries from a plant mixture comprising cedar of Lebanon and a member of the Cupressaceae family (juniper, cypress) to account for the occurrence of himachalene and cuparene derivatives. There is, to our knowledge, no historical evidence that steam distillation was a process known in Egypt at the beginning of the 3 rd millennium B.C. However, [START_REF] Levey | Some chemical apparatus of ancien Mesopotamia[END_REF] proposed that this technology was known in the Levant as early as the middle of the 4 th millennium B.C. This hypothesis relies on the discovery of a double-rimmed pot in Tepe Gawra in Iraq [START_REF] Levey | Some chemical apparatus of ancien Mesopotamia[END_REF] postulated to correspond to the oldest known part of a steam distillation apparatus. It can therefore be envisaged that preparation of the material in the jars, which most likely occurred in the Levant prior to exportation to Egypt, has been achieved using steam distillation. In such a process, the plant material mixed with water mixture is brought to boiling, and the resulting vapors comprising water and volatile organic constituents from the odoriferous plants are condensed and gathered in the trough of the double-rimmed pot, the organic phase then being separated from water and recovered.

Conclusions

GC-MS investigation of the organic materials from three jars found in Abydos and dating back to the two first Egyptian dynasties (3100-2700 B.C.) resulted in the identification of oxidized terpenic structures of the himachalene and cuparene series and totarol, which led us to propose that the organic substances were most likely prepared from at least two distinct conifer species comprising cedar of Lebanon and a member of the Cupressacea family (Cupressus sempervirens and/or one of the various juniper species growing in the Levant). The identification of novel oxidized sesquiterpenoids from the himachalene family, predominating in the severely oxised samples, was carried out with prepared reference molecules. The latter constitute the first unambiguous biomarkers related to himachalenes which can be used to identify oil from real cedar (Cedrus sp.) in organic material from ancient Egypt. The predominance of low molecular weight constituents in the extracts, together with the absence of biomarkers indicative of lignin pyrolysis suggests that the organic substances were prepared by steam distillation of conifer material rather than by dry pyrolysis of wood.

However, it cannot be completely excluded that the typical markers for tar, in particular alkyl guaiacols, were lost by evaporation or upon intense oxidative alteration during ageing.

Although there are obvious molecular analogies between the investigated material and "cedrium", we propose that the organic substances have been used as cosmetics, medicinal ingredients or for cultual purposes rather than for embalming, which is a practice that developed during later historical periods. Reinvestigation of the molecular composition of "cedrium" reported in the literature in the light of our results shows that oxidized cuparene and himachalene-related sesquiterpenoids co-occur in "cedrium", giving clues to the controversy concerning its ingredients and indicating that "cedrium" was most likely prepared using both true cedar (C. libani) and juniper or cypress. Longiborneol 1 (300 g) (respectively longicamphenilol A, 340 g) was reacted with pyridinium dichromate (20 mg) in dry CH2Cl2 (1 ml) for 2 hours at room temperature. Filtration on a short silica gel column followed by elimination of the solvent under reduced pressure yielded longicamphor 2 (respectively, longicamphenilone 3) in almost quantitative yield.

Figure captions

Longicamphor 2: GC-MS (EI, 70eV); m/z (relative intensity) 220 (M + , 92%), 177 (91), 137 The identification of compounds 1-3 in samples E21727, E21809 and 28010 was based on the comparison of mass spectra and of retention times in GC of the synthetic compounds with those of the compounds from the archaeological samples.

Identification of cuparene derivatives 5-8

Cuparene 5 was identified in the archaeological samples by comparison with the mass spectrum and retention time on GC with that of cuparene 5 from the essential oil of Texas cedar (Juniperus mexicana) (Sigma-Aldrich). The methyl ester of cuparenic acid 6 was identified based on the comparison with the mass spectrum published by Grantham and Douglas (1980).

Compounds 7 and 8 were identified based on the interpretation of their mass spectra as the analogues of compounds 5 and 6 bearing a carbonyl group on the cyclopentane ring. The mass spectra of 7 and 8 are indeed very similar to those of cuparene 5 and cuparenic acid 6 (as methyl ester), respectively, with a molecular ion shifted upwards by 14 Da and even fragments typical

Preparation and identification of himachalene-related sesquiterpenoids

Compounds 12-15, 29 and 30 were prepared from the essential oil of Cedrus atlantica (Laboratoire Sirius), the terpenoid distribution of which being dominated by β ,-α-and γhimachalene 16-18 (Fig. 6a). The experimental protocol used is shown in Fig. S5. Panday and Dev, 1968) Essential oil of Cedrus atlantica (502 mg) and chloranil (886 mg) were dissolved in toluene (10 ml). The mixture was heated at 90 °C for 4 h under an atmosphere of N2 in a tube closed with a screwcap. The solid was filtered off and washed with hexane. The crude organic mixture after filtration was fractionated by column chromatography on a silica gel column using n-hexane as eluent. Three fractions were recovered: F1a (124 mg), F2a (277 mg), F3a (24mg).

Dehydrogenation of essential oil of Cedrus atlantica with chloranil (adapted from

F2a comprised the two unsaturated analogues of ar-himachalene 29, 30 as predominant compounds and small amounts of ar-himachalene 12. Compounds 29, 30 were isolated for analytical purposes (4 mg, 3 mg, respectively) from 8 mg of F2a fraction by thin layer chromatography silica gel impregnated with silver nitrate using n-hexane/CH2Cl2 (8/2, v/v) as developer (Fig. S5). or H-5), 7.17 (3H, bs, H-2). 13 C-NMR (125 MHz; CD2Cl2): 21.4, 26.5, 31.4, 31.4, 37.8, 39.1, 41.0, 113.2, 126.7, 126.9, 130.2, 136.7, 141.2, 146.9, 154.5. 21.5, 23.7, 26.6, 31.4, 31.4, 38.3, 48.1, 126.4, 126.6, 126.7, 127.8, 136.2, 137.6, 138.3, 147.8.

Hydrogenation of unsaturated analogues (29, 30) of ar-himachalene 12

An aliquot (10 mg) of fraction F2a (cf. section 2.3.1; Fig. S5) was dissolved in EtOAc (10 ml). PtO2 (ca. 10 mg) was added and the mixture stirred under a H2 atmosphere. After 2 h, the mixture was put under N2 atmosphere and the solid catalyst was filtered off and washed with CH2Cl2. ar-Himachalene 12 was obtained in almost quantitative yield. CD2Cl2): 20.8,20.9,24.0,29.5,33.7,34.3,36.6,39.4,41.1,125.3,126.3,127.5,134.9,141.2,147.7.

Oxidation of unsaturated analogues of ar-himachalene with pyridinium chlorochromate:

Preparation of 14 and 15

An aliquot (130 mg) of fraction F2a (cf. section 2.3.1; Fig. S5) was reacted with pyridinium chlorochromate (531mg) and 5Å molecular sieves (60 beads) in dry CH2Cl2 (15 ml) under reflux. After 4 h, the reaction mixture was filtered on a small silica gel column to remove the reagents. The fraction obtained by elution with EtOAc was evaporated under reduced pressure. The crude mixture was chromatographed on a silica gel column using CH2Cl2/EtOAc, (97/3, v/v) as eluent. Seven fractions (Fig. S5) were obtained: F1b (65mg), F2b, (12mg), F3b

(11 mg) F4b (6.5 mg), F5b, (11 mg), F6b (<2 mg), F7b (7 mg). GC-MS analysis showed that 24.5,30.1, 30.4, 30.9, 33.5, 33.7, 78.8, 127.2, 127.2, 127.7, 131.3, 138.4, 146.2, 211.1.

  himachalenes 16-18, which are major constituents of essential oils from real cedars, led us to propose that the organic material originated predominantly from C. libani. The occasional occurrence of cuparene 5 in C. libani and C. atlantica[START_REF] Baser | The essential oil of Taurus cedar (Cedrus libani A. Rich): Recent results[END_REF][START_REF] Aberchane | Analysis of Moroccan Atlas cedarwood oil (Cedrus atlantica Manetti)[END_REF] in low amounts cannot account for the important proportions of cuparene derivatives 5-8 relative to other sesquiterpenoids in the extracts from the jars (cf. Fig. 2a,b and Fig S1, S2 in Supplementary data), unless considering a marked concentration effect.

Fig. 1 .

 1 Fig. 1. Jars (a) E21727, (b) E21809, (c) E28010 (©Musée du Louvre /Geneviève Pierrat-Bonnefois).

Fig. 2 .

 2 Fig. 2. Gas chromatograms of (a) the moderately polar fraction F1; (b) the fraction of the volatile compounds (F1 volatile ) isolated from the organic material Ffrom jar E21727. Alcohols are analyzed as acetates and carboxylic acids as methylesters in fraction F1. Fraction F1 volatile was not derivatized. Filled circles: linear carboxylic acids, numerals refer to the carbon number of the molecules.

Fig. 3 .

 3 Fig. 3. Mass spectra (electron impact, 70 eV) of the himachalene-related sesquiterpenoids 12-15 identified in the organic material from jars E21727, E21809 and E28010 (cf. Fig. 2, Fig. S1, Fig. S2) and of the himachalene derivatives 29-30 formed upon autoxidation of C. atlantica essential oil (cf. Fig. 6b).

Fig. 4 .

 4 Fig. 4. Possible oxidative pathway leading from terpenic structures to the aromatic (poly)carboxylic acids 22-27 found in jars E21727 and E21809.

Fig. 5 .

 5 Fig. 5. Genetic relationship between dehydrogenated and oxidized sesquiterpenoids found in in jars E21727, E21809 and E28010 and their postulated precursor molecules in the case of (a) longiborneol 1 and longifolene 4 derivatives, (b) himachalene (16-18) derivatives, (c) cuparene 5 derivatives.

Fig. 6 .Fig. 7 .

 67 Fig. 6. Gas chromatograms of (a) C. atlantica essential oil, (b) C. atlantica essential oil after oxidation with O 2 catalyzed by N-hydroxyphthalimide and cobalt(II) acetylacetonate.

Figure S1 :

 S1 Figure S1 : Gas chromatograms of the fractions F1 isolated from the organic material found in jars (a) E21809 and (b) E28010 (numbers refer to structure in the Appendix). Filled circles: fatty acids; filled triangles: 2-hydroxy acids. Alcohols were analyzed as acetates and carboxylic acids as methyl esters.

Figure S2 :

 S2 Figure S2 : Gas chromatograms of the fractions F1 volatile from the organic material found in jars (a) E21809 and (b) E28010 (numbers refer to structures in the Appendix).

  for cuparene-related compounds (m/z 132 and m/z 176, respectively, FigS4). In addition, the mass spectrum of 7 is almost superimposable with that of -cuparenone 7a from the NIST library. The position of the carbonyl group on the archaeological compounds could, however, not be determined based on the interpretation of mass spectra.

Figure S4 :

 S4 Figure S4: Mass spectra (electron impact, 70 eV) of cuparene-related sesquiterpenoids 5-8 detected in the organic material from jars E21727, E21809 and E28010

Figure S5 :

 S5 Figure S5: Preparation and isolation of himachalene-related sesquiterpenoids 12-15, 29 and 30 from Cedrus atlantica essential oil. AcCN: acetonitrile; HPLC: high performance liquid chromatography.

Compound 29 .

 29 GC-MS (EI, 70 eV); m/z (relative intensity) 200 (M + , 55%), 185 (69), 157 (100), 143 (43), 128 (13), 115 (12). 1 H-NMR (500 MHz; CD2Cl2): 1.315 (6H, s, H-14 and H-15), 1.69 (2H, m), 1.86-1.98 (2H, m), 2.31 (3H, s, H-12), 2.33 (2H, m), 4.92 (1H, d, J = 2.5 Hz, H-13), 5.03 (1H, m, H-13), 6.93 (1H, bd, J = 7.5 Hz, H-4 or H-5), 7.01 (1H, d, J = 7.5 Hz, H-4

  Compound 30 GC-MS (EI, 70 eV); m/z (relative intensity) 200 (M + , 86%),185 (100), 170 (60), 159 (80), 157 (85), 143 (45), 128 (30), 115 (18). 1 H-NMR (500 MHz; CD2Cl2): 1.300 (6H, s, H-14 and H-15), 1.88-1.96 (4H, m, H-9 and H-10), 2.09(3H, d, J = 1.5 Hz, H-13), 2.33 (3H, s, H-12), 5.86 (1H, ddd, J = 6.5 Hz, J = 6.5 Hz, J = 1.5 Hz, H-8), 7.03 (1H, bd, J = 7.5 Hz, H-4 or H-5), 7.18 (1H, d, J = 7.5 Hz, H-4 or H-5), 7.20 (3H, bs, H-2). 13 C-NMR (125 MHz; CD2Cl2):

Compound 12 .

 12 GC-MS (EI, 70 eV); m/z (relative intensity) 202 (M + , 50%), 187 (100), 159 (16), 145 (92), 131 (67), 128 (21). 1 H-NMR (500 MHz; CD2Cl2): 1.21 (1H, m), 1.312 (3H, s, H-14 or H-15), 1.319 (3H, d, J = 6.5 Hz, H-13), 1.41 (3H, s, H-14 or H-15), 1.47 (1H, m), 1.61 (1H, m), 1.72 -1.84 (3H, m), 2.28 (3H, s, H-12), 3.28 (1H, m, H-7), 6.95 (1H, d, J = 8.0 Hz, H-4 or H-5), 7.09 (1H, d, J = 8.0 Hz, H-4 or H-5), 7.17 (3H, s, H-2). 13 C-NMR (125 MHz;

fraction

  F4b contained compounds 14 and 15. Fractionation by high performance liquid chromatography (HPLC) using a Waters model 590 HPLC pump (mobile phase: n-hexane/ EtOAc 96/4, flow rate: 1 ml/min), connected to an Agilent ZORBAX-NH2 column (25 cm x 2.1 mm i.d., 5 µm) and associated to a differential refractometer detector R401 (Waters Associates) yielded compound 14 (1.8 mg) and 15 (1.1 mg). Compound 14. GC-MS (EI, 70 eV); m/z (relative intensity) 214 (M + -H2O, 3%), 199 (3), 189 (100), 171 (15), 119 (72), 105 (11), 91 (7). GC-MS (CI, isobutane) m/z (relative intensity) 233 ([M+H] + , <1%), 215 ([M+H-H2O] + , 100). 1 H-NMR (500 MHz; CD2Cl2) : 1.29 (3H, s, H-14 or H-15), 1.37 (3H, s, H-14 or H-15), 1.68 (2H, m, H-10a, H-10b), 2.02 (1H, m, H-9b), 2.04 (3H, s, H-13), 2.24 (1H, m, H-9b), 2.32 (3H, s, H-12), 4.36 (1H, s, OH), 6.68 (1H, d, J = 8.0 Hz, H-4 or H-5), 6.96 (1H, d, J = 8.0 Hz, H-4 or H-5), 7.24 (3H, s, H-2). 13 C-NMR (125 MHz; CD2Cl2): 21.1,

  and Scholz-Böttcher et al. (2013) comprised several compounds having the

	same fragmentation and molecular ions in MS as the compounds unambiguously identified for
	the first time in the present study as himachalene derivatives (compounds 12, 13, 15, 29, 30) by
	comparison with prepared standards (Section 2.3 from Supplementary data). Thus, we propose
	to reassess and/or refine the structures of some compounds reported by Koller et al. (2005) and
	Scholz-Böttcher et al. (

Acknowledgements

E. Motsch (Université de Strasbourg) is thanked for the GC-MS analyses and B. Vincent (Université de Strasbourg) for NMR measurements. J. Connan is thanked for facilitating the access to the samples. A. Charrié-Duhaut and C. Bastien (Université de Strasbourg) are thanked

for the sampling of the organic substances in jars E21727 and E21809. The "Centre National de la Recherche Scientifique" and "Université de Strasbourg" are thanked for financial support.

Supplementary

 199 (47),171 (100),157 (77),156 (98),141 (47),128 (33),115 (33). 1 H-NMR (500 MHz; CD2Cl2): 1.41 (6H, s, H-14 and H-15), 2.37 (6H, s, H-12 and H-13), 2.66 (2H, s, H-10), 6.24 (1H, s, H-8), 7.13 (1H, d, J = 8.0 Hz, H-4 or H-5), 7.27 (1H, s, H-2), 7.49 (1H, d, J = 8.0 Hz, H-4 or H-5). 13 C-NMR (125 MHz; CD2Cl2): 21.3, 21.3, 27.2, 27.2, 34.3, 56.8, 126.0, 127.0, 129.9, 130.0, 133.4, 139.8, 148.2, 150.2, 200.1.

Oxidation of compound 2 with O2 and N-hydroxyphtalimide (NHPI): Preparation of 13

(adapted from [START_REF] Lin | Metal-free, NHPI catalyzed oxidative cleavage of C-C double bond using molecular oxygen as oxidant[END_REF] Fraction F1b (cf. section 2.3.3, Fig. S5) was shown by GC-MS to be mainly constituted by the unsaturated ar-himachalene derivative 29 and minor amounts of compounds 12, 30. To an aliquot of this fraction (20 mg), NHPI (4.5 mg), H2O (2 l) and AcCN (800 l) were added and the mixture heated at 80 °C under stirring in a round-bottomed flask under O2 atmosphere for 6 hours. Because of the difficulty to handle small volumes of solvent in a round-bottom flask and to regulate the temperature, the reaction mixture was transferred into a glass tube closed with a screwcap and pursued after addition of NHPI (4.5 mg) and AcCN (1 ml) at 80 °C under O2 atmosphere. After 20 h, NHPI (4.5 mg) was added and the mixture heated for a further 8 hour time period at 80 °C under O2 atmosphere. The AcCN solution was directly extracted with pentane (4 ml, 3 x), the solvent was removed under reduced pressure and the crude extract chromatographed over a silica gel column eluting with pentane/CH2Cl2 (8/2, v/v) and CH2Cl2, successively, yielding 0.9 mg of ketone 13 (CH2Cl2 eluted-fraction; Fig. S5). (100),145 (41),144 (55),134 (36),117 (14),115 (24),91 (17),69 (14). 21.6, 21.7, 31.9, 31.9, 39.1, 40.7, 43.1, 127.1, 127.5, 128.6, 138.7, 141.2, 147.7, 208.6. Compound 24 (as trimethyl ester): GC-MS (EI, 70eV); m/z (relative intensity) 252 (M + , 7%), 222 (24), 221 (100), 193 (12), 191 (14), 162 (9), 161 (10), 103 (8) Compound 25 (as methylester): GC-MS (EI, 70eV); m/z (relative intensity) 178 (M + , 16%), 163 (100), 147 (18), 135 (27), 119 (9), 103 (10), 91 (6), 76 (6) Compound 26 (as dimethylester): GC-MS (EI, 70eV); m/z (relative intensity) 194 (M + , 26%), 179 (7), 164 (10), 163 (100), 135 (27), 120 (5), 103 (11), 76 (6)

Identification of aromatic carboxylic acids


B Löhrer

Sara Puijalon

D Doppler

N Rivière

J J S Jerome

J Fröhlich

A first simulation

Keywords: canopy, reconfiguration, high Cauchy number, water channel, scale-resolving simulation

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

INTRODUCTION

Canopies formed by aquatic plants appear abundantly in water bodies under the most diverse circumstances and play decisive roles in the respective ecosystems. Of particular relevance are canopies in flowing streams due to the mechanical interaction between the mean and instantaneous fluid velocity and the deformation of the plants undergoing substantial reconfiguration with impact on flow resistance, sediment retention, species transport, etc. [START_REF] Nepf | Flow and Transport in Regions with Aquatic Vegetation[END_REF]. Reconfiguration depends on the flow velocity and the properties of the solid bodies. It is usually expressed as a function of the Cauchy number Ca which is the ratio of the characteristic dynamic pressure of the fluid to the forces created by the stiffness of a structure. Consequently, low Cauchy numbers yield small deformation, while high Cauchy numbers entail large deformation.

To investigate fundamental issues it is convenient to consider prototypical canopies made of blades with identical shape, usually rectangular, and equal size and stiffness, together with a regular arrangement. This insures repeatability of experiments or simulations. Experiments with such canopies have been reported in several studies, such as [START_REF] Kouwen | Flexible roughness in open channels[END_REF] or [START_REF] Rominger | Effects of blade flexural rigidity on drag force and mass transfer rates in model blades[END_REF], to name but two.

Simulations of such a setting are much scarcer due to the difficulties in modeling. Statistical turbulence models including deformation have been employed [START_REF] Dijkstra | Modeling the interaction between flow and highly flexible aquatic vegetation[END_REF] Turbulence-resolving approaches, however, are very rare and more recent due to their high numerical cost. While such computations have been done in considerable number for rigid blades or beams, still demanding large computing times, the representation of fluid-structure coupling in the case of flexible blades which, in addition, also collide, constitutes a further challenge for the numericist.

In the literature few studies have been conducted with model canopies of low to moderate Ca, [START_REF] Boothroyd | The importance of accurately representing submerged vegetation morphology in the numerical prediction of complex river flow[END_REF][START_REF] Tschisgale | Simulation of the turbulent flow over an array of flexible blades[END_REF]. But to the knowledge of the authors only [START_REF] Marjoribanks | Does the canopy mixing layer model apply to highly flexible aquatic vegetation? Insights from numerical modelling[END_REF]) so far numerically simulated canopies with very high Ca, i. e. very flexible elements, in this case cylinders in a very regular staggered arrangement and with a relatively low resolution of 5 grid points per diameter.

Experiments for high-Ca canopies are difficult since the individual elements move substantially, making measurements complicated, and since, additionally, this motion obstructs the optical access to the interior of the canopy, whatever their arrangement.

The present contribution reports on a simulation of a high-Ca canopy presently mounted for experimental investigation at LMFA, Lyon. This canopy is made of long, flat ribbons of plastic with a density slightly smaller than that of water. It was devised to mimic patches of aquatic plants that are frequently formed by plants in streams [START_REF] Cornacchia | Landscapes of facilitation: How self-organized patchiness of aquatic macrophytes promotes diversity in streams[END_REF][START_REF] Licci | The role of patch size in ecosystem engineering capacity: A case study of aquatic vegetation[END_REF]). The linear strap-like shape of the blades is common among aquatic plant species (e. g. Sparganium emersum, Vallisneria spiralis) and is considered as an adaptation to flowing water [START_REF] Sand-Jensen | Drag and reconfiguration of freshwater macrophytes[END_REF][START_REF] Albayrak | Flow-plant interactions at a leaf scale: Effects of leaf shape, serration, roughness and flexural rigidity[END_REF], which makes the experimental configuration used in this study relevant to investigate interactions between hydrodynamics and plants.

NUMERICAL METHOD

Simulations were performed using the in-house code PRIME developed at TU Dresden [START_REF] Kempe | An improved immersed boundary method with direct forcing for the simulation of particle laden flows[END_REF][START_REF] Tschisgale | An immersed boundary method for the fluid-structure interaction of slender flexible structures in viscous fluid[END_REF]. With this method, the Navier-Stokes equations for incompressible flows are solved employing a second-order finite volume approach on a staggered Cartesian grid with constant step size in all directions. For the time integration a semi-implicit pressure-correction approach using a three-step, second-order Runge-Kutta method is applied. Energetic turbulent scales are resolved and sub-grid scale turbulence is accounted for via a Large Eddy Simulation (LES) approach, a subgrid-scale viscosity provided by the Smagorinsky model.

The flexible structures forming the canopy are represented by means of the geometrically exact Cosserat rod model [START_REF] Antman | Nonlinear Problems of Elasticity[END_REF] suitable for rods undergoing large deflections. Their motion is computed by a finite-difference approach according to [START_REF] Lang | Numerical aspects in the dynamic simulation of geometrically exact rods[END_REF]. Collisions are handled by the model proposed in [START_REF] Tschisgale | A constraint-based collision model for Cosserat rods[END_REF].

Coupling between fluid and structures is established with a recently developed new variant of the Immersed-Boundary Method (IBM) imposing a no-slip condition on the surface of the rods. The present implementation extends the approach of [START_REF] Tschisgale | An immersed boundary method for the fluid-structure interaction of slender flexible structures in viscous fluid[END_REF] and is applicable for small and even vanishing thickness of the blades. Details of the entire method and validations are compiled in (Tschisgale & Fröhlich, subm.).

PHYSICAL SETUP

A fully developed open-channel flow is investigated with fluid properties corresponding to those of water at a temperature of 293.15 K according to the IAPWS97 standard (see Tab. 1). A set of 132 flexible blades with the properties given in Table 2 are attached perpendicular to the bottom wall at the locations indicated in Figure 1. This pattern consists of seven unique columns in z- direction repeated in such a way that the resulting pattern cannot be subdivided into equal parts. The resulting array of blades is staggered in crosswise direction to avoid channelization. x / mm To characterize the overall problem, the following dimensionless numbers are used,

Ca = ρ U 2 b W L 3 2 EI , B = (ρ s -ρ) gW T L 3 EI , Re H = U b H ν , Re W = U b W ν , (1)
where I = T 3 W/12 is a second moment of area. The respective values for the present case are provided in Table 3. The huge Cauchy Ca and the relatively large value of |B| reflect the enormous flexibility of the blades, i. e. both, nominal drag and lift force outweigh elastic forces. The roughness density λ is defined as the ratio of the mean frontal area of a plant divided by the bed area it occupies. The latter quantity is approximated by ∆S

x ∆S z = 13.5 cm 2 , the former requires knowledge of the mean reconfigured canopy height which is a results of the simulation. The computed value provided in Table 3 satisfies λC d > 0.23, indicative of a dense canopy [START_REF] Poggi | The effect of vegetation density on canopy sub-layer turbulence[END_REF]). In addition, with the reconfigured canopy height L * ≈ 0.22H the case is in the regime of shallow submergence, H > L * > H/5 where boundary layer turbulence is usually not developed and canopy scale turbulence dominates [START_REF] Nepf | Flow and Transport in Regions with Aquatic Vegetation[END_REF].

NUMERICAL SETUP

Values for several numerical parameters are provided in the Tables 45. Periodic boundary conditions were imposed in x-direction, a rigid lid condition at the top wall, i. e. a flat geometry with free slip condition which is standard in simulations of this type [START_REF] Mcsherry | Large eddy simulation of free-surface flows[END_REF], and a no slip condition at the other three walls. The flow was driven by a spatially constant volume force adjusted in each time step to obtain the desired flow rate. This, as well, is standard for such a configuration.

While the Cosserat model accounts for bending, torsional, and shearing motion, the model assumption of constant cross section geometry prevents warping. In the present case of slender cross sections, however, warping is substantial. To remedy this issue, the effect of warping was modeled by reducing torsional and shearing stiffnesses by factors k t and k s , respectively, with corresponding values computed as proposed by [START_REF] Freund | Warping displacement of Timoshenko beam model[END_REF].

Statistics were collected after a startup time T st ≈ 46 T b , and during an averaging period T av ≈ 111 T b , where T b = H/U b ≈ 1.21 s. The time-averaged shapes of the individual blades were further averaged to obtain one mean shape. Its maximum elevation was taken as the mean reconfigured canopy height L * .

RESULTS

Figures 2 and 3 provide visualizations of the instantaneous flow for the same instant in time with the streamwise velocity component u plotted in multiple slices. The visualizations give the impression of a substantial level of turbulence ranging from small structures at the scale of the blade width to very large ones. The latter is evident from the slightly meandering streamwise streaks of large velocity with a low-velocity region in-between (Fig. 3). The blades undergo large deformations and exhibit undulations reaching up to about half the channel height with convex bending when seen from above. They also exhibit lateral motion, related to sideward inclination of their cross-section.

The profile of mean streamwise velocity u x,z,t provided in Figure 4a contains two inflection points in the canopy region which is typical for dense canopies [START_REF] Poggi | The effect of vegetation density on canopy sub-layer turbulence[END_REF]). Interestingly, though, the upper one is located at y ≈ 0.08 H ≈ 0.36L * , with L * ≈ 0.22H the mean reconfigured canopy height, whereas in flows at lower Ca it is expected to be above the averaged canopy, e. g. observed by [START_REF] Marjoribanks | Does the canopy mixing layer model apply to highly flexible aquatic vegetation? Insights from numerical modelling[END_REF]. Another significant feature of the u x,z,t profile is its maximum clearly below the surface known to be due to secondary flow structures in open-channel flows [START_REF] Nezu | Turbulence in Open-Channel Flows[END_REF]. Such a secondary flow, indeed, is seen in preliminary graphs. Streamwise fluctuations are particularly intense near the canopy top, y = L * ≈ 0.22H as can be seen from the profile of u ′ u ′ x,z,t in Figure 4c.

Although not yet quantitatively ascertained, this is expected to be due to large vertical shifts of the horizontally aligned structures separating low and high momentum flow. The maxima of -u ′ v ′ x,z,t and v ′ v ′ x,z,t coincide at a position clearly above the average canopy top. In contrast, [START_REF] Ghisalberti | Mixing layers and coherent structures in vegetated aquatic flows[END_REF] found these maxima to occur near y = L * in case of lower values of Ca, with that of -u ′ v ′ x,z,t slightly below y = L * and the other just above it. This difference is assumed to be attributed to the rather prone mean geometry of the blades. Consequently, a blade at mean reconfiguration would be lowered by a sweep only slightly, soon being hindered by neighboring plants downstream, while an ejection has the potential to simultaneously lift an entire patch of several structures.

In videos, at rare instances, the blades are observed to interpenetrate and stick together for a moment. This is a technical defect in the implementation of the collision model. Its impact on the statistics is assumed negligible, but is subject to further investigation.

CONCLUSIONS

In this study a configuration was investigated that features a streamwise homogeneous canopy of long artificial blades with high flexibility mimicking a class of patches of aquatic plants. So far, experimental data are not yet available for this configuration, but the measurements are prepared at the time of writing. This is why the simulation features lateral walls, as these are present in the experiment.

The statistical results constitute a first step towards the analysis of this flow, indicating qualitative differences from more rigid canopies. This will be continued by means of two-point correlations and spectra. Future work will also be concerned with the motion of the blades and the corresponding statistics, as well as conditional averaging to investigate features of the coupling between blades and turbulent flow.

Figure 1 .

 1 Figure 1. Pattern of locations for the fixation of flexible blades at the bottom wall, with ∆S x = 30 mm and ∆S z = 45 mm.

Figure 2 .Figure 3 .

 23 Figure 2. Instantaneous snapshot with the streamwise velocity component plotted in vertical planes. The structures are colored with a gradient (fading from root to tip) to render the individual structures more distinguishable.

Figure 4 .

 4 Figure 4. Mean streamwise velocity and Reynolds stress components, all obtained by averaging in x, z (from wall to wall) and in time.

Table 1 .

 1 Fluid properties.

							Table 2. Structure properties.
	Open water channel, H = 156 mm L z = 250 mm ρ = 998.2 kg m -3 fluid density channel height channel width ν = 1.003 • 10 -6 m 2 s -1 kin. viscosity U b = 0.129 m s -1 bulk velocity		LDPE strips under gravity g = 9.81 m/s 2 , L = 250 mm blade length W = 15 mm blade width T = 0.063 mm blade thickness ρ s = 920 kg m -3 structure density E = 250 MPa Young's modulus G = 83 MPa Shear modulus
	0	100	200	300	400	500	600	700

Table 3 .

 3 Dimensionless numbers.

	Ca = 25,000 Cauchy number
	B	= -145 Buoyancy number
	Re W = 1922 Reynolds number
	Re H = 20,064 Reynolds number
	λ	= 0.38 Roughness density

Table 4 .

 4 Num. settings of the fluid solver.

	L x =	720 mm domain length in x dir.
	N x = 1152	cells in x direction
	N y =	250	cells in y direction
	N z =	400	cells in z direction
	C S =	0.15	Smagorinsky const.
	∆t = 7 • 10 -4 s	time step size

Table 5 .

 5 Numerical settings of the structure solver.

	k s =	0.833 shear corr. factors
	k t = 7.14 • 10 -5 corr. factor for torsional warping
	N e =	108 number of elements

Acknowledgements This work was funded through the DFG-ANR project ESCaFlex. Computation time was provided by ZIH Dresden.