Blandine Courel
email: bcourel@britishmuseum.org

Pierre Adam

Philippe Schaeffer

Courel Blandine

Adam Pierre

Schaeffer Philippe

Triterpenoids as chemotaxonomic tools to identify and differentiate genuine, adulterated and archaeological balsams

Keywords: Styrax, Liquidambar, Myroxylon, GC/MS, sumaresinolic acid, 3-epi oleanolic acid

The potential of triterpenoids as chemotaxonomic tools

to identify and differentiate genuine, adulterated and archaeological balsams

Introduction

Balsams are defined as plant exudates containing a large variety of aromatic compounds, especially cinnamic and benzoic acids along with their related alcohol and ester derivatives [START_REF] Langenheim | Plant resins: chemistry, evolution, ecology, and ethnobotany[END_REF][START_REF] Custódio | True and common balsams[END_REF]. According to this definition, only the sticky resinous material collected from plant species belonging to the genera Styrax (Styracaceae), Liquidambar (Altingiaceae, formerly included within Hamamelidaceae) and Myroxylon (Fabaceae) can be considered as "true" balsams [START_REF] Langenheim | Plant resins: chemistry, evolution, ecology, and ethnobotany[END_REF][START_REF] Custódio | True and common balsams[END_REF].

The antibacterial, antifungal and expectorant properties of these substances were already known by Akkadian, Egyptian, Roman, Greek, Carthaginian, Inca and Amerindian populations.

Balsams were used to treat, among others, chronic diseases of the respiratory tract (e.g., cold and asthma) and the women pains [START_REF] Herodotus | L'enquête, books I to IV, translation[END_REF][START_REF] Hippocrates | Traité de la nature de la femme[END_REF][START_REF] Secundus | Histoire Naturelle[END_REF][START_REF] Fossey | L'infusion par la bouche dans la médecine akkadienne[END_REF][START_REF] Pauletti | The Styracaceae[END_REF][START_REF] Amigues | Le styrax et ses usages antiques[END_REF][START_REF] Hovaneissian | Analytical investigation of styrax and benzoin balsams by HPLC-PAD-fluorimetry and GC-MS[END_REF], as well as by embalmers to avoid body putrefaction [START_REF] Reutter | Archéologies, analyses de deux masses ayant servi aux Incas à embaumer leurs morts[END_REF][START_REF] Reutter | De l'embaumement chez les anciens[END_REF][START_REF] Lucas | Cosmetics, Perfumes and Incense in Ancient Egypt[END_REF]. Moreover, due to the presence of volatile aromatic compounds, balsams were used for fumigation during religious rituals or for formulation of perfumes, ointments and cosmetics [START_REF] Amigues | Le styrax et ses usages antiques[END_REF][START_REF] Chermette | Le catalogue raisonné des producteurs de styrax et d'oliban d'Edfou et d'Athribis de Haute Egypte[END_REF][START_REF] Andaya | The trans-Sumatra trade and the ethnicization of the Batak, Bijdragen tot de Taal[END_REF]. Nevertheless, the relatively similar healing and olfactory properties of the various resins known as balsams led to a major mix-up at the botanical and linguistic level. Stricto sensu, the resins collected from Styrax species (Styracaceae) should be named "styrax" whereas those obtained from Liquidambar species (Altingiaceae) should be referred to as "liquidambar" or "storax". However, this semantic distinction is not systematically made and, in the literature, the term "styrax" has been assigned interchangeably to both resins. By contrast, the denomination of Tolu balsam and Peru balsam is well defined and refers to exudates from Myroxylon balsamum and Myroxylon pereirae, respectively -or Myroxylon balsamum var. balsamum and Myroxylon balsamum var. pereirae, some botanists considering that only a single species divided into two varieties exists [START_REF] Langenheim | Plant resins: chemistry, evolution, ecology, and ethnobotany[END_REF]. Additionally, some of these resins are extremely rare because of a low yield of exudation. Such is the case for the resin from S. officinalis since the plant produces very little resinous material by notching the bark. This rarity has even led some botanists to raise serious doubts about the real existence of this balsam [START_REF] Langenheim | Plant resins: chemistry, evolution, ecology, and ethnobotany[END_REF].

Climatic and environmental conditions have a major impact on the production of the resin of S. officinalis. Thus, resin production seems to be exclusively restricted to warm climate regions. This environmental dependence for resin production was known since the Antiquity, compelling therefore Greeks to import the coveted resin from Phoenicia [START_REF] Herodotus | L'enquête, books I to IV, translation[END_REF][START_REF] Amigues | Le styrax et ses usages antiques[END_REF].

As suggested by the abundance of texts reporting on the use of balsams in the past, it is likely that organic substances containing balsams might be encountered in archaeological contexts. In this respect, balsams have indeed been identified as constituents of the organic residue found in an Egyptian censer [START_REF] Modugno | Aromatic resin characterization by gas chromatography-mass spectrometry, raw and archaeological materials[END_REF] or as an ingredient of an organic coating on a decorated skull unearthed at Nahal Hemar cave (Israel) and dated to the Pre-Pottery Neolithic B period (ca. 8200 -7300 cal. BC) [START_REF] Solazzo | Identification of the earliest collagen-and plant-based coatings from Neolithic artefacts (Nahal Hemar cave, Israel)[END_REF]. It thus appears that reliable chemotaxonomic criteria based, in particular, on lipid markers allowing unambiguous identification of archaeological balsams are desirable and have to be established.

Previous molecular studies of balsams, reviewed by Pauletti et al. [START_REF] Pauletti | The Styracaceae[END_REF] and Custódio and Veiga-Junior [START_REF] Custódio | True and common balsams[END_REF], were however mainly dedicated to fresh material and focused on the characterisation of volatile and fragrant compounds, comprising monoterpenoids and ester derivatives of cinnamic, benzoic, p-coumaric and ferulic acids in S. benzoin [START_REF] Modugno | Aromatic resin characterization by gas chromatography-mass spectrometry, raw and archaeological materials[END_REF][START_REF] Schroeder | The p-hydroxycinnamyl compounds of Siam benzoin gum[END_REF][START_REF] Popravko | Derivatives of unsaturated aromatic alcohols in propolis and styrax benzoin[END_REF][START_REF] Pastorova | Analytical study of free and esters bound benzoic and cinnamic acids of gum benzoin resins by GC-MS and HPLC-frit FAB-MS[END_REF][START_REF] Fernandez | Volatile constituents of benzoin gums: Siam and Sumatra, Part 1, Flavour Fragr[END_REF], S. paralleloneurum [START_REF] Hovaneissian | Analytical investigation of styrax and benzoin balsams by HPLC-PAD-fluorimetry and GC-MS[END_REF][START_REF] Pastorova | Analytical study of free and esters bound benzoic and cinnamic acids of gum benzoin resins by GC-MS and HPLC-frit FAB-MS[END_REF], S. tonkinensis [START_REF] Hovaneissian | Analytical investigation of styrax and benzoin balsams by HPLC-PAD-fluorimetry and GC-MS[END_REF][START_REF] Fernandez | Volatile constituents of benzoin gums: Siam and Sumatra, Part 1, Flavour Fragr[END_REF], L. styraciflua [START_REF] Fernandez | Chemical composition of the essential oils from Turkish and Honduras styrax[END_REF], L. orientalis [START_REF] Hovaneissian | Analytical investigation of styrax and benzoin balsams by HPLC-PAD-fluorimetry and GC-MS[END_REF][START_REF] Fernandez | Chemical composition of the essential oils from Turkish and Honduras styrax[END_REF][START_REF] Hafizoğlu | Chemical constituents of balsam from Liquidambar orientalis[END_REF][START_REF] Pastorova | 3-phenylpropanylcinnamate, a copolymer unit in Siegburgite fossil resin: a proposed marker for the Hammamelidaceae[END_REF] and Myroxylon pereirae [START_REF] Seo | Larvicidal activity of ajowan (Trachyspermum ammi) and Peru balsam (Myroxylon pereira) oils and blends of their constituents against mosquito, Aedes aegypti, acute toxicity on water flea, Daphnia magna, and aqueous residue[END_REF] and some specificity of the distributions of these compounds with respect to their source could be evidenced. Nevertheless, in the case of archaeological balsams, these compounds cannot be easily used to recognise the nature of the plant species since they are prone to alteration, as illustrated by the case of the balsam identified on the skull of the Nahal Hemar cave [START_REF] Solazzo | Identification of the earliest collagen-and plant-based coatings from Neolithic artefacts (Nahal Hemar cave, Israel)[END_REF]. In the latter case, the distributions of the cinnamic and benzoic acid derivatives, although useful to relate the substance on the skull to a balsam-type resin, were too altered to allow a distinction to be made between a resin from S. officinalis and L. orientalis -the two possible botanical sources in the eastern Mediterranean region -. Alteration processes which had apparently affected cinnamic and benzoic acid derivatives comprised partial loss by evaporation due to high volatility and, possibly, cross-reactions (transesterification) with other

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constituents of the organic substance on the skull [START_REF] Solazzo | Identification of the earliest collagen-and plant-based coatings from Neolithic artefacts (Nahal Hemar cave, Israel)[END_REF]. In addition, the predominant cinnamic and benzoic esters in balsams are potentially prone to hydrolysis and, similarly to unsaturated fatty acids [e.g. 25], the double bond of cinnamates might be cleaved by oxidative processes.

Therefore, other species-specific molecular tools are required for the reliable identification of balsams (e.g. Styrax sp., Liquidambar sp. or Myroxylon sp.) notably when altered material is met (e.g., during archaeological excavations).

Plant triterpenoids are potentially good candidates for this purpose since their structures present a higher resistance and lower volatility compared to the compounds mentioned above and a number of studies on resinic lipids have demonstrated the high species-specificity of some triterpenoids (e.g., [START_REF] Van Der Doelen | Comparative chromatographic and mass spectrometric studies of triterpenoid varnishes: fresh material and aged samples from paintings[END_REF][START_REF]Serpico with a contribution by R. White. Resins, amber and bitumen[END_REF][START_REF] Peters | The biomarker guide, Volume 1: Biomarkers and isotopes in the environment and human history[END_REF][START_REF] Regert | Reconstructing ancient Yemen commercial routes during the Middle Ages using structural characterization of terpenoid resins[END_REF]). In addition to the archaeological interest mentioned above, the identification of resins using triterpenes might also be useful for the detection of counterfeit balsams. As a matter of fact, triterpenes might allow not only the unambiguous correlation with the precise botanical source of the balsam to be made, but also identification of resins from other sources (e.g. dammar resin, notably) that may have been used for adulteration. Indeed, because of limited supply and high cost of some balsams, substitution resins have often been used. For instance, the gum of L. orientalis has been an alternative of the Mediterranean styrax and the resin of Altingia excelsa (Altingiaceae) was used as a substitute of styrax and liquidambar resins [START_REF] Langenheim | Plant resins: chemistry, evolution, ecology, and ethnobotany[END_REF]. The trade of imitations and adulterated products is even more problematical and appears to be a long-standing practice, Pliny the Elder reporting in the 1 st century AD some fraud cases [START_REF] Secundus | Histoire Naturelle[END_REF]. Even nowadays, the material sold on the market often turns out to be adulterated most generally with dammar resin or colophony, as reported by several authors [START_REF] Langenheim | Plant resins: chemistry, evolution, ecology, and ethnobotany[END_REF][START_REF] Wahlberg | 3-oxo-6β-hydroxyolean-12-en-28-oic acid, a new triterpenoid from commercial Tolu balsam[END_REF], and observed by ourselves during chemical analyses of commercially-available resins.

However, despite representing potential chemotaxonomic markers, there are only few studies dedicated to the triterpene composition of balsams (S. tonkinensis: [START_REF] Hovaneissian | Analytical investigation of styrax and benzoin balsams by HPLC-PAD-fluorimetry and GC-MS[END_REF][START_REF] Wang | Triterpenoids from the resin of Styrax tonkinensis and their antiproliferative and differentiation effects in human leukemia HL-60 cells[END_REF]; L. orientalis: [START_REF] Huneck | Triterpene -IV. Die Triterpensäuren des Balsams von Liquidambar Orientalis Miller[END_REF]).

We have therefore carried out a systematic and detailed molecular study of the triterpenoid

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distributions from styrax and liquidambar resins using GC/MS in order to explore their potential as chemotaxonomic indicators. For completeness, resins from different parts of the world have been investigated when they were available and unequivocally assigned to a botanical origin. Thus, in the case of Styracaceae, we have investigated two resin samples of S. officinalis native from the Middle East and the Mediterranean area [START_REF] Amigues | Le styrax et ses usages antiques[END_REF], as well as resin samples of S. tonkinensis (South Eastern Asia) and S. paralleloneurum (Malaysia and Indonesia; [START_REF] Hovaneissian | Contribution de la chimie analytique à l'étude des exsudats végétaux styrax, storax et benjoin[END_REF]). Most significantly, the lipid distribution of the resin of S. officinalis is presented here for the first time. As a representative of the Altingiaceae family, we have examined the lipid content of the gum of L. orientalis, which is endemic from Anatolia in Turkey, and that of the resin of L. styraciflua native from North and Central America [START_REF] Langenheim | Plant resins: chemistry, evolution, ecology, and ethnobotany[END_REF]. Finally, the occurrence of triterpenes was investigated within Peru balsam (M. balsamum var. pereirae) which find its origin in the Americas [START_REF] Langenheim | Plant resins: chemistry, evolution, ecology, and ethnobotany[END_REF][START_REF] Custódio | True and common balsams[END_REF]. Several plant materials (aerial parts from S. officinalis, M. balsamum var. balsamum and M. balsamum var. pereirae) were also analysed, but the outcome of the lipid analysis of these materials will only be discussed in supplementary materials as it is not the primary focus of the present paper.

Experimental

Plant and resin material

Preliminary GC/MS analysis of the lipids from a large set of commercial balsams has revealed that most of them were adulterated. Therefore, we looked insofar as possible to obtain certified exudates or plant elements (Table 1). Bark, leaves and seed samples of S. officinalis were collected in October 2014 on a shrub from the Botanical Garden of Strasbourg (France). Two fresh resin samples of S. officinalis were collected in 2015 as part of the study, one in Yodfat (Israel) kindly provided by Dr. A. Nissenbaum (Weizmann Institute of Science, Israel), Prof. Z. Ammar (Bar Ilan University, Israel), Prof. M. Kislev (Bar Ilan University, Israel) and Prof.

A. Dafni (Haifa University, Israel), the other one in the Botanical Garden of Montpellier (France) -authentication done by V. Girard (ISEM, University of Montpellier, France) -.

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Additionally, resins from S. tonkinensis ("Siam benjoin") and S. paralleloneurum ("Sumatra benjoin") were investigated (gift from E. Dodinet, Laboratoire Archéologie TRACES, University of Toulouse, France). Liquidambar resins (L. orientalis and L. styraciflua) have been purchased from the company of fragrance and aroma ROBERTET. Peru balsam was gathered in December 2016 by the company Rivera Balsam (J. Paul Rivera S.A. de C.V., El Salvador) and kindly provided for the study. Specimens of leaves and branches of M. balsamum (L.) Harms var. balsamum from Belgium (from Jardin colonial Laeken) and of M. balsamum (L.) Harms var. pereirae (Royle) Harms (from Democratic Republic of Congo, I.N.E.A.C.) were given by Dr. Marc Reynders and Mr. Franck van Caekenberghe (Botanic Garden of Meise).

Lipid extraction and sample preparation

The samples were extracted under sonication using a mixture of DCM/MeOH (1:1, v/v). An aliquot of the total lipid extract (TLE) recovered after filtration through celite and elimination of the solvents under reduced pressure was acetylated using acetic anhydride (1 mL) and Nmethylimidazole (20 µL, catalyst) for 20 min at ambient temperature. After removal of the solvents and excess reagents, the acetylated TLE was methylated with diazomethane in diethylether. Following removal of the excess of diazomethane and solvents under a gentle stream of N2, the derivatized organic extract was fractionated on a silica gel column yielding an apolar fraction eluted with a mixture of DCM/EtOAc (8:2, v/v) and a more polar fraction eluted with DCM/MeOH (1:1, v/v). The latter was not further investigated.

GC/MS

GC/MS analyses were performed on a Thermo GC Trace gas chromatograph equipped with a HP5 MS column (30 m x 0.25 mm x 0.1 µm) and a temperature programmable injector linked to a Thermo TSQ Quantum mass spectrometer. Helium was used as carrier gas and the oven was programmed as follows: 70 °C (1 min), 70 °C -200 °C (10 °C/min), 200 °C -320 °C (4 °C/min), isothermal at 320 °C for 40 min. The mass spectrometer was operating in electron Version avant révision impact mode (70 eV) with a scan range of m/z 50-700. In some cases, chemical ionisation (CI) analyses were performed using isobutane.

GC/TOF

GC-TOF analyses were carried out on a Agilent Technologies 7890A gas chromatograph equipped with a split/splitless injector (used in the splitless mode), a DB5-UI column (20 m x 0.18 mm; 0.18 µm film thickness). The GC was coupled to a time-of-flight (TOF) JEOL Accu TOF GCV JMS-T100GCV mass spectrometer operating in the EI mode (70 eV) or field ionization (FI) mode with a scan range of m/z 35-800. Helium was used as carrier gas and the temperature program was identical to that described in section 2.3.

Results

Phenylpropanoids in Styrax, Liquidambar and Myroxylon balsams

The balsams of Styrax, Liquidambar and Myroxylon species are characterised by the presence of ester derivatives of cinnamic and benzoic acids (Fig. 1, 2 and 3 respectively), this molecular assemblage being responsible for the specific pleasant flavour of the resins. In MS, detection of the ester derivatives of cinnamic and benzoic acids mainly relies on the presence of typical fragments respectively at m/z 131 and 105 (Fig. S1). However, in some cases, the mass spectra of these compounds are relatively "poor" in terms of structural information (i.e., absence of molecular ions and of diagnostic fragmentation ions) preventing unambiguous structural assignments of the alcohol moieties of benzoic acid and cinnamic esters.

Styrax

Overall, GC/MS investigation of the cinnamate and benzoate derivatives from the four styrax resins investigated in this study revealed significant differences in their molecular distributions (MS data in Supplementary Information, Fig. S1 and Table S1). Indeed, the predominance of ester derivatives of cinnamic acid vs. benzoic acid was observed in the resins of S. officinalis

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(Fig. 1a and1b) and S. paralleloneurum (Fig. 1c), whereas the opposite occurred in the exudate of S. tonkinensis (Fig. 1d).

More specifically, the two resin samples of S. officinalis are characterised by the predominance of cinnamyl cinnamate 1 (Fig. 1a and1b), followed by methoxyeugenol 2, cinnamic acid 3 and benzyl cinnamate 4 as other quantitatively important constituents. Cinnamaldehyde 5, vanillin 6, eugenol 7, vanillic acid 8, benzyl benzoate 9, cinnamyl benzoate 10, ferulic acid 11, cinnamyl alcohol 12, propyl cinnamate 13, coniferyl alcohol 14 and 3-phenylpropanyl cinnamate 15 were detected as minor constituents, compounds 12-15 being exclusively observed in the sample from Israel. The presence of 3-phenylpropanyl cinnamate 15 (trace amounts) in a Styracaceae resin may be seen as a contradiction with the statement of Pastorova et al. [START_REF] Pastorova | 3-phenylpropanylcinnamate, a copolymer unit in Siegburgite fossil resin: a proposed marker for the Hammamelidaceae[END_REF] who have suggested that this compound could be specific to the Hamamelidaceae gums. Nevertheless, when present in high relative amounts, 3-phenylpropanyl cinnamate 15 can still be considered as a chemotaxonomic marker for Hamamelidaceae gums since it is a predominant constituent of Liquidambars (see § 3.1.2 and Fig. 2). The structure of compound 16 -the latter being particularly abundant in the styrax resin from Israel and to a lesser extent in that from Francecould not be fully established although a mass fragment at m/z 167 suggests the presence of a syringyl moiety [START_REF] Hovaneissian | Contribution de la chimie analytique à l'étude des exsudats végétaux styrax, storax et benjoin[END_REF]. Compound 16 is absent from the bark and from the others aerial parts of the sample of S. officinalis investigated (see Supplementary material) and seems to be specifically associated to the resinous material.

Cinnamic acid 3 largely predominates the lipid distribution of the balsam exuded from S. paralleloneurum (Fig. 1c), in accordance with previous studies [START_REF] Langenheim | Plant resins: chemistry, evolution, ecology, and ethnobotany[END_REF][START_REF] Hovaneissian | Analytical investigation of styrax and benzoin balsams by HPLC-PAD-fluorimetry and GC-MS[END_REF]

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were detected. The rather low relative amounts (compared to free cinnamic acid) of esters of cinnamic acid (e.g., compounds 1, 4, 17 and 18) could be associated to a low-grade resin [START_REF] Pastorova | Analytical study of free and esters bound benzoic and cinnamic acids of gum benzoin resins by GC-MS and HPLC-frit FAB-MS[END_REF] or could be inherent to the resin of S. paralleloneurum.

In contrast to the resins of Styracaceae described above, the lipid extract of S. tonkinensis is characterised by abundant ester derivatives of benzoic acid (m/z 105; Fig. 1d). Indeed, coniferyl benzoate 25 represents by far the major compound in the exudate of S. tonkinensis along with vanillin 6, benzyl benzoate 9, p-coumaric acid 23 and p-coumaryl benzoate 24 occurring as minor constituents [START_REF] Hovaneissian | Analytical investigation of styrax and benzoin balsams by HPLC-PAD-fluorimetry and GC-MS[END_REF][START_REF] Popravko | Derivatives of unsaturated aromatic alcohols in propolis and styrax benzoin[END_REF]. The importance of 25 was already noticed by Hovaneissian et al. [START_REF] Hovaneissian | Analytical investigation of styrax and benzoin balsams by HPLC-PAD-fluorimetry and GC-MS[END_REF] who proposed to use this compound as a marker for Siam benzoin. GC/MS analysis revealed small quantities of compounds 2-4, 7, 8, 10, 11, 14, 19 and 20, also found in S. officinalis and S. paralleloneurum (with the exception of 19 and 20 for the latter resin), together with the benzoate derivatives 27 to 29.

Liquidambars

The chromatographic profiles corresponding to the apolar lipids of the gums from L. orientalis and L. styraciflua (Fig. 2) unveiled substantial similarities and clearly differ from those of the resins of Styracaceae by the strong predominance of cinnamyl cinnamate 1 and 3phenylpropanyl cinnamate 15. Although trace amounts of 15 were detected in some styrax resins (see above), its high abundance together with that of 1 is a typical feature of freshly collected resins from liquidambars as shown by Pastorova et al. [START_REF] Pastorova | 3-phenylpropanylcinnamate, a copolymer unit in Siegburgite fossil resin: a proposed marker for the Hammamelidaceae[END_REF]. Among the minor compounds, the presence of cinnamic acid 3, benzyl cinnamate 4, cinnamyl alcohol 12, pcoumaryl cinnamate 17 and 3-phenylpropanol 30 in both liquidambars was observed. Other compounds were identified exclusively in the American liquidambar (L. styraciflua), namely cinnamyl benzoate 10, p-coumaryl alcohol 22 and 3-(4-hydroxyphenyl)-propyl cinnamate 26.

Apart from a few exceptions such as that of benzyl benzoate 9 not detected in our liquidambar gums, the molecular distributions of cinnamates and benzoates are close to those described in the literature for liquidambar exudates [START_REF] Hovaneissian | Analytical investigation of styrax and benzoin balsams by HPLC-PAD-fluorimetry and GC-MS[END_REF][START_REF] Hafizoğlu | Chemical constituents of balsam from Liquidambar orientalis[END_REF][START_REF] Pastorova | 3-phenylpropanylcinnamate, a copolymer unit in Siegburgite fossil resin: a proposed marker for the Hammamelidaceae[END_REF][START_REF] Acar | Investigation of chemical composition of natural Liquidambar orientalis Mill balsam by gas chromatography-mass spectrometer combined system, Doga[END_REF].

Peru balsam (Myroxylon balsamum (L.) Harms var. Pereirae)

Ester derivatives of cinnamic and benzoic acids were also identified in Peru balsam (Fig. 3).

Benzyl benzoate 9 prevails in the lipid extract -as already observed in a 18 th century Peru balsam from the Vigani Cabinet [START_REF] Steigenberger | The Vigani Cabinet -Analysis of historical resinous materials by gas chromatography -mass spectrometry and infrared spectroscopy[END_REF] -and cinnamic acid 3 with benzyl cinnamate 4 figure among the main constituents. Compounds 31 to 33 were also detected in a relatively large amount in the lipid extract of Peru balsam, but their structures were difficult to establish based solely on GC/MS data (MS shown in Fig. S2). Compounds 31 and 33 are likely to be phenylpropanoid derivatives as they show characteristic MS base peaks of benzoate and cinnamate derivatives (m/z 105 and m/z 131, respectively). Similarly, 32 is characterised by a main fragment at m/z 91 as commonly observed for aromatic compounds. However, being out of scope of our study which primarily aims at looking for triterpenoid markers, no further structural investigation was carried out. Finally, in accordance with the work of Seo et al. [START_REF] Seo | Larvicidal activity of ajowan (Trachyspermum ammi) and Peru balsam (Myroxylon pereira) oils and blends of their constituents against mosquito, Aedes aegypti, acute toxicity on water flea, Daphnia magna, and aqueous residue[END_REF], E-nerolidol was also identified in significant amount.

Lignan and neolignan derivatives in aromatic balsams

Investigation of the two resins of S. officinalis revealed the occurrence of the neolignans egonol 34 and homoegonol 35 belonging to the benzofuran group (EI mass spectra shown in Supplementary, Fig. S3). The relative abundance of 34 and 35 in the sample from Israel was particularly marked (Fig. 1b) and has likely been induced by unintentional addition of small bark fragments during resin collection. This was further confirmed by the occurrence of 34 and 35 as predominant neolignans in the bark from S. officinalis (Fig. S4a). The same compounds were also found in the fruits from the plant (Fig. S4c), together with related derivatives 36-40, the structure of which being proposed based on the interpretation of their mass spectra. It is noteworthy that 34 and 35 were also identified in trace amount in the resin of S. paralleloneurum and S. tonkinensis (Fig. 1). According to the literature, it appears more broadly that egonol and homoegonol are found in aerial parts (leaves, stems, seeds, bark) of trees from the genus Styrax ( [START_REF] Pauletti | The Styracaceae[END_REF] and references herein, [START_REF] Moraes | Development and validation of a high-performance liquid chromatography method for quantification of egonol and homoegonol in Styrax species[END_REF][START_REF] De Oliveira | Study of the cytotoxic activity of Styrax camporum extract and its chemical markers, egonol and homoegonol[END_REF][START_REF] Timmers | Characterization of phenolic compounds and antioxidant and antiinflammatory activities from Mamuyo (Styrax ramirezii Greenm.) fruit[END_REF]).

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Lignans are constituted of two monolignol units (e.g., coniferyl alcohol) linked at the carbon position C8-C8' (examples of EI mass spectra are shown in Supplementary, Fig. S5). Besides pinoresinol 41 ubiquitous in the Plant Kingdom [START_REF] Milder | Lignan contents of Dutch plant foods: a database including lariciresinol, pinoresinol, secoisolariciresinol and matairesinol[END_REF][START_REF] Durazzo | Phenols, lignans and antioxidant properties of legume and sweet chestnut flours[END_REF], the two resins from S. officinalis contain styraxin 42 (also occurring in the bark as shown in Fig. S4a), sesamin 43 and piperitol 44 (Fig. 1a and1b). As far as we know, since its characterisation by Ulubelen et al. [START_REF] Ulubelen | Chemical components of Styrax officinalis L. IV. The structure of a new lignan styraxin[END_REF] in the aerial parts of S. officinalis, styraxin 42 has not been reported to occur in other Styrax species and in any other plants. Hence, styraxin 42 could represent a specific marker of S. officinalis and could be used as an indicator for the use of S. officinalis resin in an archaeological context, as attested by its identification in an 8000 years-old decorated skull [START_REF] Solazzo | Identification of the earliest collagen-and plant-based coatings from Neolithic artefacts (Nahal Hemar cave, Israel)[END_REF].

Aside from pinoresinol 41 which was detected in the twigs from M. balsamum var. balsamum (Fig. S6), none of the lignans and neolignans described above was identified in the liquidambar gums, in the Peru balsam and the other aerial parts of the Myroxylon species investigated in this study.

Triterpenoids in styrax resins

There are only a few reports in the literature on the triterpenoid content of Styracaceae. These include the triterpenoids from the stem-bark of S. japonica [START_REF] Kwon | Triterpenoids and a sterol from the stem-bark of Styrax japonica and their protein tyrosine phosphatase 1B inhibitory activities[END_REF], from the leaves of S. camporum [START_REF] Pauletti | Triterpenos de Styrax camporum (Styracaceae)[END_REF], and, surprisingly, there is only one triterpenoid study on the resin of a member of the genus Styrax (S. tonkinensis; [START_REF] Wang | Triterpenoids from the resin of Styrax tonkinensis and their antiproliferative and differentiation effects in human leukemia HL-60 cells[END_REF]). The triterpenoid content of the resins from S. officinalis and S. paralleloneurum have not been investigated to date, with the exception of the article by Yayla et al. [START_REF] Yayla | Saponins from Styrax officinalis[END_REF] reporting triterpene saponins from S. officinalis. This limited number of studies might be partly explained by their very low abundance relative to cinnamate and benzoate derivatives in the resins. GC/MS investigation of the triterpenoids from S. officinalis, S. paralleloneurum and S. tonkinensis resin showed that they belong to the oleanane, and, to a lesser extent, to the ursane family (Fig. 4a), and can be divided into three group series:

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(a) triterpenoids relatively common in the Plant Kingdom, namely oleanonic [START_REF] Yayla | Saponins from Styrax officinalis[END_REF], oleanolic [START_REF] Budzikiewicz | Mass spectrometry in structural and stereochemical problems. XXXII. 1. Pentacyclic triterpenes[END_REF] and ursolic (47) acids (EI mass spectra shown in Supplementary; Fig. S7a-c) (b) rather rare triterpenoids having the same main fragments in MS as 45-47, but with molecular ions shifted upwards by 14 or 16 mass units (48-50; Fig. S7d-f).

(c) uncommon triterpenoids displaying a base fragment at m/z 201 and a minor fragment at m/z 278 in their mass spectra (51 and 52; Fig. S7g-h).

For the compounds from the second series, the absence or very low intensity of molecular ions under the conditions used (triple quadrupole, EI, 70 eV) initially prevented us to propose a generic structure. However, GC-HRMS analysis of the same compounds using a TOF mass analyser under either EI or FI allowed the molecular mass of 48 (M+ . 484), 49 (M+ . 526) and 50 (M+ . 528) to be unambiguously determined. These data, in combination with the fragmentation pattern observed, are in good agreement with the mass spectra of triterpenoids belonging to the sumaresinolic acid generic series reported in a commercial Tolu balsam (Myroxylon balsamum) by Wahlberg and Enzell [START_REF] Wahlberg | 3-oxo-6β-hydroxyolean-12-en-28-oic acid, a new triterpenoid from commercial Tolu balsam[END_REF]. It should however be mentioned that the exact nature of this commercial Tolu balsam has to be questioned, as noted by the authors themselves, and it is likely that this "Tolu" balsam was in fact a counterfeit made with a mixture of resins of different botanical origins comprising Dipterocarpaceae resin (see discussion below in paragraph 4). The EI mass spectra of compounds 48 (3-oxo-sumaresinolic acid), 49 and 50 (sumaresinolic acid) are shown in Supplementary Fig. S7d-f. In agreement with the data from Wahlberg and Enzell [START_REF] Wahlberg | 3-oxo-6β-hydroxyolean-12-en-28-oic acid, a new triterpenoid from commercial Tolu balsam[END_REF], the lack of reactivity of the hydroxyl group at C-6 towards acetylation confirmed that it is located at a sterically-hindered position (i.e., at the axial position).

The uncommon triterpenoids of the third series (51 and 52) belong to the oleanane series as well and were exclusively detected in the resin of S. tonkinensis. The presence of a fragment at m/z 278 in their mass spectra might be ascribed to a retro-Diels-Alder type fragmentation of the

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ring C of a triterpenoid having a Δ 12 double bond and a carbomethoxy group at C-17 (m/z 262; [START_REF] Budzikiewicz | Mass spectrometry in structural and stereochemical problems. XXXII. 1. Pentacyclic triterpenes[END_REF]) plus a hydroxyl group on the C/D/E ring moiety, as further confirmed by the loss of H2O (m/z 260) from the m/z 278 fragment (EI mass spectra and fragmentation pattern shown in Supplementary; Fig. S7g-h). The fact that this hydroxyl group could not be acetylated suggests that it is located at C-19 at a sterically-hindered position (i.e., α stereochemistry) [START_REF] Schnell | Contrasting diagenetic pathways of higher plant triterpenoids in buried wood as a function of tree species[END_REF]. The mass spectrum of 51 (Fig. S7g; Supplementary Information) is in agreement with published data [START_REF] Jakupovic | Sesquiterpene glycosides and other constituents from Osteospermum species[END_REF].

For compound 52, the molecular ion could not be observed under EI (Fig. S7h, Supplementary Information), but was unambiguously present using CI with isobutane as reactant gas ([M+H] + at m/z 529). It can thus be proposed that compound 52 represents the C-3 acetylated derivative of 3β,19α-dihydroxy-olean-12-en-28-oic acid (siaresinolic acid).

Triterpenoids in liquidambar gums

The triterpenoid distributions of the two liquidambar gums are closely related (Fig. 4b) and are characterised by the presence of derivatives of oleanolic and ursolic acids (presence of typical fragments at m/z 203 and m/z 262; [START_REF] Budzikiewicz | Mass spectrometry in structural and stereochemical problems. XXXII. 1. Pentacyclic triterpenes[END_REF]). They comprise oleanonic acid 45 and its 3-epi isomer 53, as described by Huneck [START_REF] Huneck | Triterpene -IV. Die Triterpensäuren des Balsams von Liquidambar Orientalis Miller[END_REF], along with oleanolic acid 46, maslinic acid 54, ursonic acid 55 and 3-epi-ursolic acid 56. Additionally, 6β-hydroxy-3-oxo-olean-12-en-28-oic acid 48 (3oxo-sumaresinolic acid) was detected, this compound being reported to occur in the cones of L. styraciflua [START_REF] Fukuda | Lupane and oleanane triterpenoids from the cones of Liquidambar styraciflua[END_REF]. Furthermore, oleanolic and oleanonic aldehydes 57 and 58 bearing the aldehyde group at C-28, as shown in MS by the occurrence of a retro-Diels-Alder type fragment at m/z 232, were detected. Finally, constituents from the lupane series comprising lupeol 59 and 6β-hydroxy-3-oxo-lup-20(29)-en-28-oic acid 60 were identified. The latter has also been reported to occur in the cones from L. styraciflua [START_REF] Fukuda | Lupane and oleanane triterpenoids from the cones of Liquidambar styraciflua[END_REF][START_REF] Sakai | New cytotoxic oleanane-type triterpenoids from the cones of Liquidambar styraciflua[END_REF].

Cinnamate and benzoate derivatives versus triterpenoids as lipid markers to differentiate between balsams

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Fresh styrax resins, liquidambars and Peru balsam can be differentiated by their content in phenylpropanoids as summarized in Table 2. Cinnamyl cinnamate 1, cinnamic acid 3 and coniferyl benzoate 25 represent the main constituents of the resin from S. officinalis, S. paralleloneurum and S. tonkinensis respectively whereas cinnamyl cinnamate 1 with 3phenylpropanyl cinnamate 15 prevail in both L. orientalis and L. styraciflua gums. By consequence, in the case of fresh, undegraded material, each of those compounds described above can be regarded as specific biomarkers allowing a distinction to be made between styrax and liquidambar resins as well as within the Styrax species. Furthermore, the distribution of ester derivatives of cinnamic acid and benzoic acid in the Peru balsam, dominated by benzyl cinnamate 4 and benzyl benzoate 9, also clearly differs from those observed in the styrax and liquidambar resins. However, given their structures, these aromatic derivatives are sensitive towards alteration processes and their genuine distributions will likely be lost and/or strongly modified in the case of severely altered material, as is generally the case with archaeological findings. Furthermore, the lower molecular weight homologues (e.g., free cinnamic and benzoic acids) will be lost preferentially over time because of their high volatility and water solubility (leaching). Additionally, the relative distribution of the aromatic ester derivatives can depend upon the quality of the resins [START_REF] Pastorova | Analytical study of free and esters bound benzoic and cinnamic acids of gum benzoin resins by GC-MS and HPLC-frit FAB-MS[END_REF], which hinders quantitative analysis from being used as a discriminating tool. Intermolecular esterification reactions may also occur during ageing processes, leading to the neoformation of cinnamate and benzoate derivatives absent from fresh balsams. For instance, such a possibility may explain the presence of a number of high molecular weight ester derivatives of cinnamic and benzoic acids detected in a 8000-years old organic coating from a decorated human skull [START_REF] Solazzo | Identification of the earliest collagen-and plant-based coatings from Neolithic artefacts (Nahal Hemar cave, Israel)[END_REF]. Following these considerations, ester derivatives of cinnamic and benzoic acids on their own are not reliable markers to allow a clear discrimination to be made between balsams, notably in the case of ancient material unearthed at archaeological sites.

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For this reason, triterpenoids were investigated in order to evaluate their potential as speciesspecific biomarkers. The styrax resins studied are all characterised by the occurrence of sumaresinolic acid 50 and its derivatives (48 and 49; Fig. 4a), in accordance with the molecular description of a S. tonkinensis resin reported by Wang et al. [START_REF] Wang | Triterpenoids from the resin of Styrax tonkinensis and their antiproliferative and differentiation effects in human leukemia HL-60 cells[END_REF]. In addition, the composition of the resin of S. tonkinensis contrasts with other styrax resins by the presence of siaresinolic acid 51 and its C3-ketone analogue 52. Despite a thorough search for the presence of compounds 50 and 52, they could not be detected in the other balsams investigated, which led us to explore their potential as taxonomic markers for styrax resins.

According to the literature, it appears that sumaresinolic and siaresinolic acids are not restricted to the Styrax genus but also occur in the aerial parts of plant species from other genera (e.g., [START_REF] Ming-An | Triterpenoid saponins from the leaves of Ilex latifolia[END_REF][START_REF] Cunha | In vitro trypanocidal activity of triterpenes from Miconia species[END_REF][START_REF] Garcez | Chemical constituents from Terminalia glabrescens[END_REF][START_REF] Rauter | A new lupene triterpenetriol and anticholinesterase activity of Salvia sclareoides[END_REF][START_REF] Estrada | Chemical constituents from Chrysobalanus venezuelanus[END_REF][START_REF] De Oliveira | Antinociceptive and anti-inflammatory activity of the siaresinolic acid, a triterpene isolated from the leaves of Sabicea grisea Cham. & Schltdl. var. grisea[END_REF]). Nonetheless, none of these plants produce resinous exudates containing ester derivatives of cinnamic and benzoic acids. Certainly, 3-oxo-sumaresinolic acid 48 was identified in the cones of L. styraciflua [START_REF] Fukuda | Lupane and oleanane triterpenoids from the cones of Liquidambar styraciflua[END_REF][START_REF] Fukuda | Cancer chemopreventive activity of lupane-and oleanane-type triterpenoids from the cones of Liquidambar styraciflua[END_REF] and minute amount of 48 was also found within the two liquidambar balsams investigated (Fig. 4b), but not in association with 49 and 50.

Compounds 48 and 50 were reported by Wahlberg and Enzell in a commercial Tolu balsam as well [START_REF] Wahlberg | 3-oxo-6β-hydroxyolean-12-en-28-oic acid, a new triterpenoid from commercial Tolu balsam[END_REF][START_REF] Wahlberg | Constituents of commercial Tolu Balsam[END_REF] and in collection material [START_REF] Steigenberger | The Vigani Cabinet -Analysis of historical resinous materials by gas chromatography -mass spectrometry and infrared spectroscopy[END_REF]. However, the identification of dammarane skeletons including dammaradienone, 20R-hydroxy-dammarenone and 20S-dammarenediol [START_REF] Steigenberger | The Vigani Cabinet -Analysis of historical resinous materials by gas chromatography -mass spectrometry and infrared spectroscopy[END_REF][START_REF] Wahlberg | Constituents of commercial Tolu Balsam[END_REF] within the "Tolu balsam" triterpenoids suggests that these Tolu balsams investigated were adulterated by resinous material from Dipterocarpaceae (dammar resin; [START_REF] Van Der Doelen | Analysis of fresh triterpenoid resins and aged triterpenoid varnishes by high-performance liquid chromatography-atmospheric pressure chemical ionisation (tandem) mass spectrometry[END_REF][START_REF] Lampert | Characterization and radiocarbon dating of archaeological resins from Southeast Asia[END_REF]) -as envisaged by the authors themselves [START_REF] Wahlberg | 3-oxo-6β-hydroxyolean-12-en-28-oic acid, a new triterpenoid from commercial Tolu balsam[END_REF][START_REF] Steigenberger | The Vigani Cabinet -Analysis of historical resinous materials by gas chromatography -mass spectrometry and infrared spectroscopy[END_REF] -which consequently brings into question the origin of sumaresinolic acid in the balsams. Unfortunately, we did not manage to obtain certified Tolu balsam from Myroxylon balsamum var. balsamum to carry out our own investigation but GC-MS analysis of certified Peru balsam (M. balsamum var. peirerae) from El Salvador attested to the complete absence of triterpenoids within this exudate. It is also worth noting that sumaresinolic derivatives 48-50 could not be detected in the aerial part of S. officinalis (Fig. S4), suggesting that these compounds are inherent to the resinous material.

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Hence, we suggest that the co-occurrence of the triterpenoids 48-50 together with ester derivatives of cinnamic and benzoic acids could be considered as characteristic of resins from Styrax species. Furthermore, among the resins investigated, only Styrax species did contain the neolignans 34 (egonol) and 35 (homoegonol), in agreement with the literature ( [START_REF] Pauletti | The Styracaceae[END_REF] and references herein, [START_REF] Moraes | Development and validation of a high-performance liquid chromatography method for quantification of egonol and homoegonol in Styrax species[END_REF][START_REF] De Oliveira | Study of the cytotoxic activity of Styrax camporum extract and its chemical markers, egonol and homoegonol[END_REF][START_REF] Timmers | Characterization of phenolic compounds and antioxidant and antiinflammatory activities from Mamuyo (Styrax ramirezii Greenm.) fruit[END_REF]). Because of that, they can be considered as additional specific molecules which may be used to recognise styrax resins. It is however important to note that 34 and 35 are typical constituents not only of styrax resin, but also of the aerial parts of the plants (bark and seeds; Fig. S4a,c). Therefore, their presence within the resin lipids may be attributed to the contribution of bark, for example, that could have been collected while harvesting the resin.

Liquidambar resins are characterised by a high abundance of oleanonic acid 45 along with a low amount of oleanolic and ursolic acids having the uncommon 3α-OH stereochemistry (53 and 56). This triterpenoid assemblage can be defined as specific to liquidambars since it has not been observed with the balsams of other botanical origin.

The present study shows that triterpenoids represent powerful taxonomic tools for the characterisation of higher plant resins, allowing a clear distinction between styrax resins and liquidambar gums to be made using molecular criteria. The Peru balsam investigated was characterised by a complete lack of triterpenoids which questions the findings from previous studies of the literature on the triterpenic content of Peru and Tolu balsam [START_REF] Wahlberg | 3-oxo-6β-hydroxyolean-12-en-28-oic acid, a new triterpenoid from commercial Tolu balsam[END_REF][START_REF] Wahlberg | Constituents of commercial Tolu Balsam[END_REF]. Finally, the lipid content of balsams from Myroxylon would need to be carefully investigated given the presence of numerous (and sometimes predominant) compounds with uncharacterised structures.

The research has been funded by a PhD grant given by The French Ministry of Higher Education and Research.

Myroxylon balsams

Peru balsam

Benzyl benzoate 9 --Table 2 Molecular criteria for discriminating between styrax resins, liquidambar resins and Peru balsam. acid 63 (Fig. S4). It is worth noting that the seeds were devoid of triterpenoids and comprised predominantly lignin/neolignan derivatives (see above; Fig. S4c).

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Triterpenoids in plant material from Myroxylon species

No triterpenes were detected by GC/MS in the Peru balsam (Myroxylon balsamum var.

Pereirae) whereas the apolar lipid fractions obtained from the aerial parts of M. balsamum var.

balsamum and M. balsamum var. pereirae (Fig. S6) have revealed the presence of various triterpenoids in leaves and branches along with other lipids comprising sesquiterpenoids, sugars, phenolic compounds, fatty acids, isoflavonoids and phytosteroids. The branches and leaves are notably characterised by the presence of lupeol 59, β-amyrin 64 and 11-oxo-β-amyrin 65 (Fig. S6a andS6b). In addition, an unusual oleanane derivative bearing a methoxy function at C-11 (66) and a diene (67) which could be a degradation product of 66 was exclusively observed in the leaves. Based on a previous study carried out on triterpenoids from the leaves of M. balsamum [START_REF] Langenheim | Plant resins: chemistry, evolution, ecology, and ethnobotany[END_REF], an α-stereochemistry for the methoxy group at C-11 could be suggested for 67. Furthermore, the C-2,3 hydroxylated triterpenoids 68-71 were also reported to occur in the leaves [START_REF] Langenheim | Plant resins: chemistry, evolution, ecology, and ethnobotany[END_REF]. No. Compound name MW Characteristic fragments (EI, m/z, %) 1 cinnamyl cinnamate 264 77 [START_REF] Pastorova | 3-phenylpropanylcinnamate, a copolymer unit in Siegburgite fossil resin: a proposed marker for the Hammamelidaceae[END_REF], 91 [START_REF] Solazzo | Identification of the earliest collagen-and plant-based coatings from Neolithic artefacts (Nahal Hemar cave, Israel)[END_REF], 103 [START_REF] Łucejko | Characterisation of archaeological waterlogged wood by pyrolytic and mass spectrometric techniques[END_REF], 115 [START_REF] Pauletti | Triterpenos de Styrax camporum (Styracaceae)[END_REF], 116 [START_REF] Reutter | Archéologies, analyses de deux masses ayant servi aux Incas à embaumer leurs morts[END_REF], 117 [START_REF] Regert | Reconstructing ancient Yemen commercial routes during the Middle Ages using structural characterization of terpenoid resins[END_REF], 131 (100), 132 [START_REF] Reutter | De l'embaumement chez les anciens[END_REF], 219 [START_REF] Pastorova | 3-phenylpropanylcinnamate, a copolymer unit in Siegburgite fossil resin: a proposed marker for the Hammamelidaceae[END_REF], 264 (3) 2 methoxyeugenol 236 91 [START_REF] Hovaneissian | Analytical investigation of styrax and benzoin balsams by HPLC-PAD-fluorimetry and GC-MS[END_REF], 103 [START_REF] Popravko | Derivatives of unsaturated aromatic alcohols in propolis and styrax benzoin[END_REF], 119 [START_REF] Amigues | Le styrax et ses usages antiques[END_REF], 131 (66), 147 [START_REF] Amigues | Le styrax et ses usages antiques[END_REF], 162 [START_REF]Serpico with a contribution by R. White. Resins, amber and bitumen[END_REF], 163 [START_REF] Acar | Investigation of chemical composition of natural Liquidambar orientalis Mill balsam by gas chromatography-mass spectrometer combined system, Doga[END_REF], 167 [START_REF] Timmers | Characterization of phenolic compounds and antioxidant and antiinflammatory activities from Mamuyo (Styrax ramirezii Greenm.) fruit[END_REF], 193 [START_REF] Hovaneissian | Analytical investigation of styrax and benzoin balsams by HPLC-PAD-fluorimetry and GC-MS[END_REF], 194 (100), 195 [START_REF] Lucas | Cosmetics, Perfumes and Incense in Ancient Egypt[END_REF], 236 (9) 3 cinnamic acid (Me) 162 51 [START_REF] Lucas | Cosmetics, Perfumes and Incense in Ancient Egypt[END_REF], 77 [START_REF] Wang | Triterpenoids from the resin of Styrax tonkinensis and their antiproliferative and differentiation effects in human leukemia HL-60 cells[END_REF], 102 [START_REF] Modugno | Aromatic resin characterization by gas chromatography-mass spectrometry, raw and archaeological materials[END_REF], 103 (72), 131 (100), 132 [START_REF] Reutter | Archéologies, analyses de deux masses ayant servi aux Incas à embaumer leurs morts[END_REF], 161 [START_REF] Acar | Investigation of chemical composition of natural Liquidambar orientalis Mill balsam by gas chromatography-mass spectrometer combined system, Doga[END_REF], 162 (60) 4 benzyl cinnamate 238 51 [START_REF] Pauletti | The Styracaceae[END_REF], 65 [START_REF] Chermette | Le catalogue raisonné des producteurs de styrax et d'oliban d'Edfou et d'Athribis de Haute Egypte[END_REF], 77 [START_REF] Wahlberg | 3-oxo-6β-hydroxyolean-12-en-28-oic acid, a new triterpenoid from commercial Tolu balsam[END_REF], 89 [START_REF] Fossey | L'infusion par la bouche dans la médecine akkadienne[END_REF], 91 (73), 92 [START_REF] Pauletti | The Styracaceae[END_REF], 102 [START_REF] Pauletti | The Styracaceae[END_REF], 103 [START_REF] Yayla | Saponins from Styrax officinalis[END_REF], 104 [START_REF] Chermette | Le catalogue raisonné des producteurs de styrax et d'oliban d'Edfou et d'Athribis de Haute Egypte[END_REF], 115 [START_REF] Van Der Doelen | Comparative chromatographic and mass spectrometric studies of triterpenoid varnishes: fresh material and aged samples from paintings[END_REF], 116 [START_REF] Secundus | Histoire Naturelle[END_REF], 131 (92), 132 [START_REF] Reutter | De l'embaumement chez les anciens[END_REF], 147 [START_REF] Pauletti | The Styracaceae[END_REF], 178 [START_REF] Solazzo | Identification of the earliest collagen-and plant-based coatings from Neolithic artefacts (Nahal Hemar cave, Israel)[END_REF], 179 [START_REF] Fossey | L'infusion par la bouche dans la médecine akkadienne[END_REF], 191 [START_REF] Pauletti | The Styracaceae[END_REF], 192 (100), 193 (68), 194 [START_REF] Hovaneissian | Analytical investigation of styrax and benzoin balsams by HPLC-PAD-fluorimetry and GC-MS[END_REF], 220 [START_REF] Amigues | Le styrax et ses usages antiques[END_REF], 238 (12) 5 cinnamaldehyde 132 77 [START_REF] Van Der Doelen | Comparative chromatographic and mass spectrometric studies of triterpenoid varnishes: fresh material and aged samples from paintings[END_REF], 78 [START_REF] Pastorova | 3-phenylpropanylcinnamate, a copolymer unit in Siegburgite fossil resin: a proposed marker for the Hammamelidaceae[END_REF], 103 [START_REF] Pauletti | Triterpenos de Styrax camporum (Styracaceae)[END_REF], 104 [START_REF] Fernandez | Volatile constituents of benzoin gums: Siam and Sumatra, Part 1, Flavour Fragr[END_REF], 131 (100), 132 (49) 6 vanillin (Ac) 194 81 [START_REF] Secundus | Histoire Naturelle[END_REF], 109 [START_REF] Pauletti | The Styracaceae[END_REF], 123 [START_REF] Reutter | Archéologies, analyses de deux masses ayant servi aux Incas à embaumer leurs morts[END_REF], 151 (85), 152 (100), 194 (9) 7 eugenol (Ac) 206 55 (4), 77 [START_REF] Amigues | Le styrax et ses usages antiques[END_REF], 91 [START_REF] Lucas | Cosmetics, Perfumes and Incense in Ancient Egypt[END_REF], 104 [START_REF] Reutter | De l'embaumement chez les anciens[END_REF], 131 [START_REF] Chermette | Le catalogue raisonné des producteurs de styrax et d'oliban d'Edfou et d'Athribis de Haute Egypte[END_REF], 133 [START_REF] Lucas | Cosmetics, Perfumes and Incense in Ancient Egypt[END_REF], 149 [START_REF] Seo | Larvicidal activity of ajowan (Trachyspermum ammi) and Peru balsam (Myroxylon pereira) oils and blends of their constituents against mosquito, Aedes aegypti, acute toxicity on water flea, Daphnia magna, and aqueous residue[END_REF], 164 (100), 165 [START_REF] Reutter | Archéologies, analyses de deux masses ayant servi aux Incas à embaumer leurs morts[END_REF] [START_REF] Fernandez | Volatile constituents of benzoin gums: Siam and Sumatra, Part 1, Flavour Fragr[END_REF], 53 [START_REF] Andaya | The trans-Sumatra trade and the ethnicization of the Batak, Bijdragen tot de Taal[END_REF], 79 [START_REF] Schroeder | The p-hydroxycinnamyl compounds of Siam benzoin gum[END_REF], 89 [START_REF] Solazzo | Identification of the earliest collagen-and plant-based coatings from Neolithic artefacts (Nahal Hemar cave, Israel)[END_REF], 91 [START_REF] De Oliveira | Study of the cytotoxic activity of Styrax camporum extract and its chemical markers, egonol and homoegonol[END_REF], 97 [START_REF] Reutter | De l'embaumement chez les anciens[END_REF], 105 [START_REF] Pastorova | 3-phenylpropanylcinnamate, a copolymer unit in Siegburgite fossil resin: a proposed marker for the Hammamelidaceae[END_REF], 124 [START_REF] Reutter | De l'embaumement chez les anciens[END_REF], 135 [START_REF] Reutter | De l'embaumement chez les anciens[END_REF], 136 [START_REF] Reutter | Archéologies, analyses de deux masses ayant servi aux Incas à embaumer leurs morts[END_REF], 138 [START_REF] Reutter | Archéologies, analyses de deux masses ayant servi aux Incas à embaumer leurs morts[END_REF], 145 [START_REF] Ming-An | Triterpenoid saponins from the leaves of Ilex latifolia[END_REF], 147 [START_REF] Wang | Triterpenoids from the resin of Styrax tonkinensis and their antiproliferative and differentiation effects in human leukemia HL-60 cells[END_REF], 150 [START_REF] Reutter | Archéologies, analyses de deux masses ayant servi aux Incas à embaumer leurs morts[END_REF], 151 [START_REF] Wahlberg | Constituents of commercial Tolu Balsam[END_REF], 152 [START_REF] Solazzo | Identification of the earliest collagen-and plant-based coatings from Neolithic artefacts (Nahal Hemar cave, Israel)[END_REF], 153 [START_REF] Schroeder | The p-hydroxycinnamyl compounds of Siam benzoin gum[END_REF], 162 (86), 163 [START_REF] Hovaneissian | Contribution de la chimie analytique à l'étude des exsudats végétaux styrax, storax et benjoin[END_REF], 175 [START_REF] Chermette | Le catalogue raisonné des producteurs de styrax et d'oliban d'Edfou et d'Athribis de Haute Egypte[END_REF], 177 [START_REF] Wahlberg | Constituents of commercial Tolu Balsam[END_REF], 179 [START_REF] Lucas | Cosmetics, Perfumes and Incense in Ancient Egypt[END_REF], 193 [START_REF] Seo | Larvicidal activity of ajowan (Trachyspermum ammi) and Peru balsam (Myroxylon pereira) oils and blends of their constituents against mosquito, Aedes aegypti, acute toxicity on water flea, Daphnia magna, and aqueous residue[END_REF], 194 [START_REF] Budzikiewicz | Mass spectrometry in structural and stereochemical problems. XXXII. 1. Pentacyclic triterpenes[END_REF], 195 [START_REF] Fernandez | Volatile constituents of benzoin gums: Siam and Sumatra, Part 1, Flavour Fragr[END_REF], 207 [START_REF] Reutter | Archéologies, analyses de deux masses ayant servi aux Incas à embaumer leurs morts[END_REF] [START_REF] Chermette | Le catalogue raisonné des producteurs de styrax et d'oliban d'Edfou et d'Athribis de Haute Egypte[END_REF], 103 [START_REF] Andaya | The trans-Sumatra trade and the ethnicization of the Batak, Bijdragen tot de Taal[END_REF], 119 [START_REF] Popravko | Derivatives of unsaturated aromatic alcohols in propolis and styrax benzoin[END_REF], 124 [START_REF] Modugno | Aromatic resin characterization by gas chromatography-mass spectrometry, raw and archaeological materials[END_REF], 131 [START_REF] De Oliveira | Study of the cytotoxic activity of Styrax camporum extract and its chemical markers, egonol and homoegonol[END_REF], 147 [START_REF] Modugno | Aromatic resin characterization by gas chromatography-mass spectrometry, raw and archaeological materials[END_REF], 151 [START_REF] Popravko | Derivatives of unsaturated aromatic alcohols in propolis and styrax benzoin[END_REF], 162 [START_REF] Lucas | Cosmetics, Perfumes and Incense in Ancient Egypt[END_REF], 163 [START_REF] Modugno | Aromatic resin characterization by gas chromatography-mass spectrometry, raw and archaeological materials[END_REF], 179 [START_REF] Lampert | Characterization and radiocarbon dating of archaeological resins from Southeast Asia[END_REF], 180 [START_REF] Hafizoğlu | Chemical constituents of balsam from Liquidambar orientalis[END_REF], 222 (100), 223 [START_REF] Lucas | Cosmetics, Perfumes and Incense in Ancient Egypt[END_REF], 264 (13) 15

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Version avant révision

3-phenylpropanyl cinnamate 266 51 [START_REF] Secundus | Histoire Naturelle[END_REF], 77 [START_REF] Van Der Doelen | Comparative chromatographic and mass spectrometric studies of triterpenoid varnishes: fresh material and aged samples from paintings[END_REF], 91 [START_REF] Huneck | Triterpene -IV. Die Triterpensäuren des Balsams von Liquidambar Orientalis Miller[END_REF], 102 [START_REF] Popravko | Derivatives of unsaturated aromatic alcohols in propolis and styrax benzoin[END_REF], 103 [START_REF] Timmers | Characterization of phenolic compounds and antioxidant and antiinflammatory activities from Mamuyo (Styrax ramirezii Greenm.) fruit[END_REF], 115 [START_REF] Reutter | Archéologies, analyses de deux masses ayant servi aux Incas à embaumer leurs morts[END_REF], 117 (95), 118 (100), 119 [START_REF] Chermette | Le catalogue raisonné des producteurs de styrax et d'oliban d'Edfou et d'Athribis de Haute Egypte[END_REF], 131 [START_REF] Hovaneissian | Contribution de la chimie analytique à l'étude des exsudats végétaux styrax, storax et benjoin[END_REF], 147 (6)

16

Unknown structure 400 ? 103 (4), 131 (9), 151 [START_REF] Fossey | L'infusion par la bouche dans la médecine akkadienne[END_REF], 152 [START_REF] Pauletti | The Styracaceae[END_REF], 167 (100), 209 [START_REF] Van Der Doelen | Comparative chromatographic and mass spectrometric studies of triterpenoid varnishes: fresh material and aged samples from paintings[END_REF], 323 (1), 340 (2), 400 (< 1) 17 p-coumaryl cinnamate (Ac) 322 77 [START_REF] Reutter | De l'embaumement chez les anciens[END_REF], 103 [START_REF] Fernandez | Chemical composition of the essential oils from Turkish and Honduras styrax[END_REF], 105 [START_REF] Amigues | Le styrax et ses usages antiques[END_REF], 131 (100), 132 [START_REF] Lucas | Cosmetics, Perfumes and Incense in Ancient Egypt[END_REF], 133 [START_REF] Modugno | Aromatic resin characterization by gas chromatography-mass spectrometry, raw and archaeological materials[END_REF], 149 [START_REF] Secundus | Histoire Naturelle[END_REF], 235 [START_REF] Reutter | De l'embaumement chez les anciens[END_REF], 280 ( 16)

18
coniferyl cinnamate (Ac) 352 77 [START_REF] Amigues | Le styrax et ses usages antiques[END_REF], 103 [START_REF] Seo | Larvicidal activity of ajowan (Trachyspermum ammi) and Peru balsam (Myroxylon pereira) oils and blends of their constituents against mosquito, Aedes aegypti, acute toxicity on water flea, Daphnia magna, and aqueous residue[END_REF], 131 (100), 132 [START_REF] Reutter | Archéologies, analyses de deux masses ayant servi aux Incas à embaumer leurs morts[END_REF], 179 [START_REF] Reutter | Archéologies, analyses de deux masses ayant servi aux Incas à embaumer leurs morts[END_REF], 265 [START_REF] Modugno | Aromatic resin characterization by gas chromatography-mass spectrometry, raw and archaeological materials[END_REF], 310 [START_REF] Pastorova | 3-phenylpropanylcinnamate, a copolymer unit in Siegburgite fossil resin: a proposed marker for the Hammamelidaceae[END_REF] [START_REF] Reutter | Archéologies, analyses de deux masses ayant servi aux Incas à embaumer leurs morts[END_REF], 91 [START_REF] Reutter | Archéologies, analyses de deux masses ayant servi aux Incas à embaumer leurs morts[END_REF], 93 [START_REF] Reutter | Archéologies, analyses de deux masses ayant servi aux Incas à embaumer leurs morts[END_REF], 105 [START_REF] Seo | Larvicidal activity of ajowan (Trachyspermum ammi) and Peru balsam (Myroxylon pereira) oils and blends of their constituents against mosquito, Aedes aegypti, acute toxicity on water flea, Daphnia magna, and aqueous residue[END_REF], 121 (100), 152 [START_REF] Van Der Doelen | Analysis of fresh triterpenoid resins and aged triterpenoid varnishes by high-performance liquid chromatography-atmospheric pressure chemical ionisation (tandem) mass spectrometry[END_REF], 162 [START_REF] Reutter | Archéologies, analyses de deux masses ayant servi aux Incas à embaumer leurs morts[END_REF], 194 (13)

22

p-coumaryl alcohol (Ac) 234 65 [START_REF] Reutter | De l'embaumement chez les anciens[END_REF], 77 [START_REF] Fernandez | Volatile constituents of benzoin gums: Siam and Sumatra, Part 1, Flavour Fragr[END_REF], 91 [START_REF] Fernandez | Volatile constituents of benzoin gums: Siam and Sumatra, Part 1, Flavour Fragr[END_REF], 94 [START_REF] Peters | The biomarker guide, Volume 1: Biomarkers and isotopes in the environment and human history[END_REF], 103 [START_REF] Colombini | Characterisation of organic residues in pottery vessels of the Roman age from Antinoe (Egypt)[END_REF], 104 [START_REF] Lucas | Cosmetics, Perfumes and Incense in Ancient Egypt[END_REF], 105 [START_REF] Milder | Lignan contents of Dutch plant foods: a database including lariciresinol, pinoresinol, secoisolariciresinol and matairesinol[END_REF], 107 [START_REF] Łucejko | Characterisation of archaeological waterlogged wood by pyrolytic and mass spectrometric techniques[END_REF], 115 [START_REF] Seo | Larvicidal activity of ajowan (Trachyspermum ammi) and Peru balsam (Myroxylon pereira) oils and blends of their constituents against mosquito, Aedes aegypti, acute toxicity on water flea, Daphnia magna, and aqueous residue[END_REF], 121 [START_REF] Schnell | Contrasting diagenetic pathways of higher plant triterpenoids in buried wood as a function of tree species[END_REF], 131 [START_REF] Sakai | New cytotoxic oleanane-type triterpenoids from the cones of Liquidambar styraciflua[END_REF], 132 [START_REF] Peters | The biomarker guide, Volume 1: Biomarkers and isotopes in the environment and human history[END_REF]

Figure caption Figure 1 .

 caption1 Figure caption

Figure 2 .

 2 Figure 2. Gas chromatograms (TIC) of an apolar fraction isolated from the lipid extract

Figure 3 .

 3 Figure 3. Gas chromatograms (TIC) of an apolar fraction isolated from the lipid extract of Peru

Figure 4 .

 4 Figure 4. m/z 189+201+203 mass fragmentograms showing the triterpenoids content a) in the

Fig. 1

 1 Fig. 1

Fig. 2

 2 Fig. 2

Fig. 3

 3 Fig. 3

Fig. 4 Investigation of the triterpenoid content of plant materials 1 .

 41 Fig. 4

[ 1 ]

 1 L. Mathias, I.J.C. Vieira, R. Braz-Filho, E. Rodrigues Filho, A new pentacyclic triterpene isolated from Myroxylon balsamum (syn. Myroxylon peruiferum), J. Brazil. Chem.Soc. 11 (2000) 195-198.

Fig S2 .

 S2 Fig S2. Mass spectra (EI, 70 eV) of major compounds detected in Peru balsam. Compounds 31-33: unknown structures.

Figure S3 .

 S3 Figure S3. Mass spectra (EI, 70 eV) of egonol 34 and homoegonol 35 as acetate derivatives.

Figure S4 .

 S4 Figure S4. Gas chromatograms of an apolar fraction isolated from the lipid extract obtained from aerial parts of S. officinalis: a) bark; b) leaves; c) fruits (Botanical Garden of Strasbourg, France). Alcohols and carboxylic acids are analyzed as acetates and methyl esters, respectively.

Figure S5 .

 S5 Figure S5. Mass spectra (EI, 70 eV) of pinoresinol 41 and styraxin 42 as acetate derivatives.

Figure S6 .

 S6 Figure S6. Gas chromatograms of the apolar fraction isolated from lipid extracts of a) branches of M. balsamum var. balsamum; b) branches of M. balsamum var. pereirae; c) leaves of M. balsamum var. balsamum; d) leaves of M. balsamum var. pereirae. Alcohols and carboxylic acids are analysed as acetates and methyl esters, respectively.

Figure S7 .

 S7 Figure S7. Mass spectra (EI, 70 eV) of acetate and/or methyl ester derivatives from triterpenic biomarkers of styrax resins: a) oleanonic acid 45; b) oleanolic acid 46; c) ursolic acid 47; d) 3oxo-sumaresinolic acid 48; e) 3b-hydroxy-6-oxo-olean-12-en-28-oic acid 49; f) sumaresinolic acid 50; g) 3-oxo-siaresinolic acid 51; h) siaresinolic acid 52.

Figure S8 .

 S8 Figure S8. Lipids identified in the resins investigated in the present study and in the aerial parts of S. officinalis, M. balsamum var. balsamum and M. balsamum var. pereirae.

-11, 14

  . It is associated with pcoumaryl cinnamate 17, cinnamyl cinnamate 1, benzyl cinnamate 4 and coniferyl cinnamate 18(in decreasing order of relative abundance). In addition to minor amounts of compounds 5

and 15, also present in the resin of S. officinalis, small amounts of 4-hydroxybenzaldehyde 19, chavicol 20, 4-hydroxybenzoic acid 21, p-coumaryl alcohol 22, p-coumaric acid 23, coumaryl benzoate 24, coniferyl benzoate 25 and 3-(4-hydroxyphenyl)-propyl cinnamate 26

Table 1

 1 Sample list of resins and aerial parts from Styrax, Liquidambar and Myroxylon species.

		Version avant révision
		Sample	Plant element	Provenance
		number
	Styracaceae, Styrax sp.
	S. officinalis	2982	resin (+ bark)	Yodfat, Israel, collected in 2015
		3020	resin	Botanic Garden of Montpellier, France,
				collected in 2015
		-	fruits	Botanic Garden Strasbourg, France, collected
		-	leaves	in October 2014
		-	bark
	S. parallelon	2984	resin	Sumatra (Indonesia)
	S. tonkinensis	2986	resin	Bought in Syria
	Altingiaceae (formerly included within Hamamelidaceae), Liquidambar sp.
	L. orientalis	-	gum	ROBERTET
	L. styraciflua	-	gum	ROBERTET
	Fabaceae, Myroxylon sp.
	M. balsamum	19373049-F	leaves	Botanic Garden of Meise (Belgium) -source :
	var. balsamum	19373049-B	branches	Jardin colonial Laeken, Bruxelles (Belgium)
	M. balsamum	-	Peru balsam	J. Paul Rivera S.A. de C.V., El Salvador,
	var. pereirae			date of production: December 2016
		19546864-F	leaves	Botanic Garden of Meise (Belgique) -
		19546864-B	branches	source : Institut National pour l'Etude
				Agronomique du Congo Belge, Democratic
				Republic of Congo

Present address: The British Museum, Great Russell Street, London WC1B 3DG, United Kingdom * Corresponding authors.E-mail addresses : BCourel@britishmuseum.org (B. Courel), p.schaef@unistra.fr (P. Schaeffer).

[START_REF] Modugno | Aromatic resin characterization by gas chromatography-mass spectrometry, raw and archaeological materials[END_REF], 91[START_REF] Fossey | L'infusion par la bouche dans la médecine akkadienne[END_REF], 103[START_REF] Hovaneissian | Analytical investigation of styrax and benzoin balsams by HPLC-PAD-fluorimetry and GC-MS[END_REF], 105 (100), 119[START_REF] Fossey | L'infusion par la bouche dans la médecine akkadienne[END_REF], 131[START_REF] Popravko | Derivatives of unsaturated aromatic alcohols in propolis and styrax benzoin[END_REF], 162[START_REF] Secundus | Histoire Naturelle[END_REF], 179[START_REF] Garcez | Chemical constituents from Terminalia glabrescens[END_REF], 284 (64), 285[START_REF] Reutter | De l'embaumement chez les anciens[END_REF], 326[START_REF] Pauletti | The Styracaceae[END_REF]

Acknowledgments

We are grateful to Dr. A. Nissenbaum (Weizmann Institute of Science, Israel), Prof. Z. Ammar (Bar Ilan University), Prof. M.Kislev (Bar Ilan University) and Prof. A. Dafni (Haifa University) for providing the resin of S. officinalis collected in Israel. We also thank Dr. J. Connan (Pau, France), Dr. V. Girard (University of Montpellier, France), Mr. F. Tournay (Botanical Garden of Strasbourg, France), Mrs. E. Dodinet (University of Toulouse, France), Pr. A. Lobstein (Pharmacy Faculty, Strasbourg, France), Dr. F. Bonté (LVMH Research, France), Dr. M. Reynders (Botanical Garden of Meise, Belgium) and Mr. F. Van Caekenberghe (Botanical Garden of Meise, Belgium) for providing fresh samples of resins and plant materials.

José Roberto Rivera is thanked for the Peru balsam that he kindly offered (company Rivera Balsam, J. Paul Rivera S.A. de C.V., El Salvador). Mr. Patrick Wehrung and Ms. Estelle Mostch are greatly thanked for their assistance in GC-TOF and GC-MS analysis, respectively.

Appendix

Funding

Version avant révision

Conflict of interest

The authors declare that they have no conflict of interest.

Supplementary Information