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1. Introduction        The study of archaeological resins can give        information relative to the man-environment relationship, the        ethnology and technological knowledge of civilizations. In the case of        Aztecs, these information are highly valuable because of the lack of        documents on pre-Columbian period.        Between 2006 and 2008 archaeological excavations        took place at Templo Mayor Site, and a resinous material attracted the        attention of archaeologists and conservation professionals, because of        their abundant presence in offerings, their versatile use, but also of        the diversity on conservation states related to degradation, involving        changes in color and surface texture. About offerings context, copal        was used as molding material for figurines, spiritual offering and        adhesive. To our knowledge formulations related to different uses have        never been studied before and the botanical origin of the employed        resins is a major concern within archaeobotanic research field.                  Historical background and copal in ancient          Mexico          The knowledge of art crafts manufacturing in          Ancient Mexico is quite ambiguous and most of the time is          fragmentary and scarce, due to the lack of documentary sources. This          research focuses on the study of two amorphous artifacts, collected          from offerings 120 and 126 at Templo Mayor. Table 1 provides          additional information about these materials.          Table 1: Archaeological samples          informationTableau 1 : Informations sur les          échantillons archéologiques                    The “Templo Mayor” was built on the geographical          center of Mexico City. It was a precinct surrounded by a staggered          platform, on which the main buildings of the city where located. It          was the tributary center of the Empire. Merchandises from a vast          territory either sold in markets or paid as tribute (Carrasco,          1999), were put together in offerings and funerary deposits. In this          temple, took place all ritual celebrations: from the regular ones,          marked in preColumbian calendar to the most extraordinary ones          corresponding to the enthronization and funerary ceremonies for the          emperor (Lopez-Luján and Chavez, 2010). In each of these events          offerings were buried (Matos-Moctezuma, 2011). When Aztecs arrived          to Mexico Valley the Tecpanecs from Azcapotzalco dominated the          region. After a short period they established in Chapultepec from          which they were expelled. Then they settled in Tizapan a territory          dominated by Culhuacan, but they left this settlement because of the          hostile environment. The Mexicas or Aztecs were an ethnic group with          nahua filiation. After a long peregrination, they funded          Mexico-Tenochtitlan, a city that between the XVth and XVIth          centuries became the center of one of the biggest empires in          Mesoamerica. Aztecs developed one of the richest and more complex          religious, political, cosmologic, astronomic, philosophic and          artistic traditions.          The offerings found in the Templo Mayor of          Tenochtitlan are made by sets of objects carefully ordered in layers          (López-Lújan, 1993). Some of these were placed in stone boxes          underneath the buildings, whereas others were buried directly in the          constructive filling materials between constructive stages of Templo          Mayor pyramid. Among the elements gotten back from these offerings          were: figurines representing deities, scepters, a pectoral, masks,          lappets, flutes, whistles, drums, instruments of sacrifice, a wooden          mask, flora and fauna remains, etc (López-Lújan and Chavez, 2010).          The first Spanish people on American continent kept the Mexican term          “copal” as well as the term “incienso de indias” (incense from          India) to refer to the natural resins, produced by New World trees,          that were burnt during religious ceremonies, probably because of the          parallel use in Europe of Boswellia resins          (frankincense). Mesoamerican cultures considered copal as a          protector. The representation of copal can often be found in codex          where priest are offering it to the gods. In goldsmith, copal was          used as wax is used in nowadays, in the “lost wax” technique. It was          part of the formulas that were used as adhesives for masks, small          rocks, and even in dentistry where it was used in dental          incrustations, in teeth cavities and in dental restorations as well          (Bucio et al, 2006).                          Natural plant resins: botanic and chemical          composition          To our knowledge few researches deal with the          chemical composition of mexican copal. Some articles describe          Mexican commercial resins (De-la-Cruz-Cañizares et          al., 2005; Orta-Amaro, 2007; Vandenabeele et          al., 2007; Hernández-Vázquez et al.,          2010;) and it is possible to find papers based on the study of          archaeological objects (Magar and Menar,1995; Stacey and Cartwright,          2006 and Vandenabeele, 2003). They respectively analyzed a turquoise          mosaic, a series of objects from the British Museum and a figure          molded in resin. Finally a recent study from our research team          (Lucero-Gómez, 2012, Lucero-Gómez et al.,          2014) focused only on the study of the triterpenic composition of          certified origin resins.          Mexican copal resins are produced by trees that          belong to the genus Bursera spp. from          Burseraceae family. Currently, in Mexico, there are more than 80          species of resin producing Bursera trees          (Rzendowski et al., 2005). Bursera species are highly resinous and release a          characteristic pine-lemony odor when a leaf or branch is broken. The          smell is due to highly volatile terpene compounds such as α-pinene,          β-phelandrene, limonene, carene, etc. (Becerra et          al., 2001). Oleoresins are referred to as “oilyresins” when          they are very fluid. This fluidity is due to a high ratio of          volatile to non-volatile terpenes (Langenheim, 2003).          In Burseraraceae, triterpenoids are the primary          components of the non-volatile fraction.          It is important to note that some other          substances can be intermixed or mistaken with resins, among which          gums can be mentioned (Langenheim, 2003). Chemically gums are          composed of complex chains of hydrophilic polysaccharides derived          from monosaccharide molecules such as galactose, arabinose, and          rhamnose. Whistler (1993) described in detail the very complex          structure of exudated gums.          Given the large number of possible botanical          origins for historical samples, the purpose of this research was to          establish molecular profiles that allow the discrimination of          botanical origin of samples, so it was necessary to analyze resins          from botanically-certified origin. The choice of the botanical          species was based on specialized literature and more precisely on          previous works about Mexican copal (Magaloni et          al., 1993; Stacey and Cartwright, 2006; Montúfar, 2007;          Hernández-Vázquez et al,.2010) to increase          the possibilities of achieving our goal. Six species were selected          for this study: B. bipinnata, B. stenophylla, B. simaruba, B.          excelsa, B. laxiflora and B.          penicillata.          The difficulty of plant resins analyses relies on          the fact that no general analytical technique exists to characterize          the molecules entering in their composition and their degradation          products (Colombini et al., 2000). The second          difficulty in analyzing these materials by HPLC relies on the nature          of the resins themselves, which are chemically very complex (Witte          et al., 1986). Indeed, Bursera resins consist of a wide variety of          compounds with very similar structures. This molecular complexity          increases with degradation process. Additionally, analysis of          archaeological samples is difficult because of the restrictions on          the number of samples and their size.                          Chemometrical Approach          HPLC-UV/Vis is a reasonably sensitive analytical          tool that allows molecular profiling. In this research, this          technique was used coupled to two multistatistical methods PCA          (Principal Component Analysis) and LDA (Linear Discriminant          Analysis).          A number of papers have identified natural          materials by means of chemometric approaches (Tapp et al., 2003; Rezzi et          al., 2005; Armanino et al., 2008; Casale          et al., 2010a, Casale et          al.,2010b). The most common is PCA (Principal Component          Analysis), which has been applied to the discrimination of          proteinaceous media (Colombini et al., 1999;          Andreoti et al., 2006; ; Bonaduce et al., 2006; Gautier and Colombini, 2007),          drying oils (Andreoti et al., 2006; Colombini          et al, 1999), polysaccharides (Colombini et al, 2002) and resins (Vieillescazes et al, 1996).          In the present work, this approach allowed a          better discrimination of chromatographic profiles in order to          enhance interpretation of analytical results. A description of the          statistical tools employed can be found in the methods section of          this work.                      2. Material and        methods        A protocol using HPLC (High Performance Liquid        Chromatography) was employed in this research. Chromatographic        techniques are powerful tools applied in several laboratories        specialized in artworks because of their ability to separate organic        compounds from complex mixtures (Doménech-Carbó, 2008).                  Statistical Methods: Principal Component          Analysis (PCA)          The aim of this method is to capture the          intrinsic variability in the data and at the same time, to reduce          the dimensionality of a data set, either to ease interpretation or          as a way to avoid overfitting and to prepare for further analysis          (Terradez, 2000). In this way PCA succeeds in explaining the maximum          amount of variance with the fewest number of main components (the          smallest loss of information). Principal components are linear          combinations of the original variables with the first principal          component having the largest variance, the second having the second          largest variance and so on (Cosio et al,          2007). PCA also allows considering the preferred directions of the          data and ignoring the variables that do not have a big influence in          the separation of the groups (Barreto, 2009). When data is projected          in two dimensions (or three), it is possible to distinguish visually          the different classes that could appear, grouped in the projection.          The different classes will be formed by those observations that          display an uniform behaviour to each other and differ from other          existing groups (García, 2002).                          Statistical Methods: Linear discriminant          analysis (LDA)          LDA is a powerful chemometric tool that allows          finding a predictive classification model, meaning that it allows          the construction of a model able to foretell the property of a          sample according to a previously defined category, or to investigate          the way variables contribute to the separation in categories. This          means that it allows studying the degree in which different          populations, established a priori, differ          from each other. It deals with two kinds of problems: descriptive          discrimination, in which it describes how two (or more) populations          differ from each other; and classification in strict sense, in which          there are two or more given populations and an object that can          belong to one of them. The discriminating analysis requires to          obtain a linear (or nonlinear) combinations of independent variables          that will discriminate between defined groups a          priori, so that the errors of the wrong classification must be          minimum (Barreto, 2009).          The LDA model is based on a set of samples of          well-known category, called training data set, and then a regression          equation is used as discriminating function (Casale et al, 2010a).          Once the model is obtained, it is used for the          prediction of the category of property of the samples of unknown          category. LDA can be linear or quadratic. The linear model assumes          that all the groups have the same covariance matrix, while quadratic          ones do not make this assumption.          In this research a level-one-out procedure was          used. This means that each sample was once removed from the data          set. The classification model is rebuilt and the removed sample is          classified in this new model. All the samples of the data set are          sequentially removed and reclassified. Finally a percentage of the          correct classification is calculated (Lachlan, 1992). The quality of          the LDA classification model was modeled according to the basis of          the validation results. The statistic analysis (PCA and LDA) in this          work was performed using Minitab 16 (Minitab Inc. PA,          California).                          Reagents          Standard molecules were used for their          identification in molecular composition of archaeological resins:          α-amyrin, β-amyrin, lupeol, and lupenone were purchased from          Extrasynthèse (Genay, France), while α-amyrone and β-amyrone were          purchased from BCP instruments (Irigny, France), 3-epi-lupeol, 3-epi-          α-amyrin, 3-epi-β-amyrin were isolated by our          team from commercial resin samples (Culioli et          al, 2003).                          Samples of resin from botanical certified          origin          Certified resin samples from six species of Bursera trees were collected in Mexican          territory. The choice of these particular species was defined by two          factors: previous research results (Stacey and Cartwright 2006;          Montúfar, 2007 and Hernández-Vázquez 2010) and to obtain an overview          on the variability of the chemical composition of Bursera resins from Mexico. Moreover, to rule out          different molecular compositions of resin from the same species,          samples were taken from different trees.          For the collection of the certified-origin          samples, this research was realized with the invaluable          collaboration of experts in botanic (Dr. Rito Vega) and in          biomaterials (Dr. Irma Belio) from the Autonomous University of          Sinaloa (UAS), as well as with the cooperation of the academic team          of Biomaterials from the UAS (UAS CA-208). The collection was          realized from summer to fall, period during trees have both leaves          and fruits, which are necessary for botanical identification. After          practicing a small cut into tree barks, resin was collected in glass          tubes. In table 2, information relating the number of samples for          each species and their geographical location (GPS position) are          presented.          Table 2: Number of certified samples studied          and their geographical locationTableau 2 :          Nombre d’échantillons certifiés étudiés et leur emplacement          géographique                                                                          Botanical species                                                                                      Number of collected samples                                                                                      Date of collection                    (dd/mm/yyyy)                                                                                      GPS localization                                                                                                              B. bipinnata                                                                                      11                                                                                      09/08/2010                                                                                      25°07’21.42’N                                                                                      107°11’57.42’W                                                                                                               B. excelsa                                                                                      4                                                                                      05/09/2009                                                                                      24°50’37.62’N                                                                                      107°02’28.76’W                                                                                                              B. laxiflora                                                                                      14                                                                                      01/11/2009                                                                                      24°45’36.06’N                                                                                      107°11’51.48’W                                                                                                              B. penicillata                                                                                      6                                                                                      29/08/2009                                                                                      24°46’08.64’N                                                                                      107°09’28.26’W                                                                                                              B. simaruba                                                                                      15                                                                                      01/11/2009                                                                                      24°51’27.60’N                                                                                      107°01’07.02’W                                                                                                              B. stenophylla                                                                                      8                                                                                      19/09/2009                                                                                      26°41’26.40’N                                                                                      108°19’58.70’W                                                                                  Historical samples           The two archaeological samples come from          offerings discovered at Templo Mayor Site. Sample referred “A” comes          from offering 120 while sample “B” comes from offering 126, both          offerings were unearthed in a lot where once stood a colonial          building known as “Casa de las Ajaracas”. The principal stairs of          the sixth constructive stage of the Templo Mayor were built in the          same physical space that “Casa de las Ajaracas” (Matos, 2007).                          Gum analysis          The aim ofthese analyses is to evaluate the gum          percentage in the raw resinous material. The gum part is          hydrosoluble and the resinous part is soluble in methanol. This          study was performed solving a known amount of fresh resin in          methanol. After sonication and centrifugation, the residue left in          the bottom of the solution was dried and weighed. The percentage of          gum was calculated from this data. Finally, water was added to          ensure the polarity of the residue and to verify its total          solubility.                          Analytical conditions in HPLC-UV/Vis           For the HPLC study, both standards and global          resins were solved in methanol (analytical grade, Merck). The          protocol consists in solubilizing an amount of crude resin (variable          depending on botanical origin) in 2 mL of this solvent, then the          solution was sonicated during 10 min. After centrifugation, the          supernatant was injected into the HPLC system. Each sample was          injected in triplicate to ensure reproducibility of the results.          Analyses were performed using a reverse phase          column with an elution gradient. The type of column used in this          research field is essential, taking into account the previous          results obtained by our team. Indeed, these works (Mathe et al, 2004), showed that for resins, a column in          reverse phase C18 led to satisfactory separations and resolutions of          the compounds. This study was thus carried out using a column with          non-polar stationary phase Merck Lichrocart Purostar 5 μm 100 RP-18e          250 mm x 4 mm (Merck, Darmstadt, Germany).          LC-PDA analysis was carried out in a Waters          liquid chromatograph consisting of a high-pressure ternary pump          Waters 600, a vacuum degasser, a high pressure manual injection          valve (20 µL injection loop) and a photodiode array detection (PDA)          system Waters 2996. The system was controlled by Empower 2 software.          The LC separation was performed at 35° C with a mobile phase          consisting of a binary elution composed of (A) bidistilled water          containing 0.01% trifluoroacetic acid (TFA) and (B) methanol          analytical grade (Merck). The chromatograms were acquired at 210 nm.          Identification of original triterpenoids was based on the          co-injection of samples with reference molecules. Chromatographic          analyses of standard molecules, certified resins and archaeological          sample A were carried out for 35 min at a continuous flow-rate of 1          mL/min. For the study of archeological sample B, a prolonged          gradient was necessary as compounds that were not present in fresh          samples were detected. The gradient profile is presented in table          3.          Table 3: HPLC gradient profil.Tableau 3 : Gradient CLHP.                                                                          Time (min)                                                                                      0                                                                                      10                                                                                      15                                                                                      35                                                        a                                                                                      45                                                        b                                                                                                              % Methanol                                                                                      85                                                                                      95                                                                                      100                                                                                      100                                                                                      100                                                                                                              % H                                                        2                                                        O-TFA                                                                                      15                                                                                      5                                                                                      0                                                                                      0                                                                                      0                                                                                                              Flow : 1 mL.min                                                        -1                                                                                      Temperature of the column = 35                    °C                                                                  a Final run time for standard molecules,          certified resins and archaeological sample A; b          Final run time for sample Ba Temps final          d’analyse pour les molécules standards, les résines certifiées et          l’échantillon archéologique A ; b Temps final d’analyse pour l’échantillon          archéologique B                      3. Results of HPLC-UV/Vis study                  Standard molecules          Liquid chromatography was applied to the study of          six standard molecules (table 4) at 210 nm, which is the general          wavelength number for the study of resins (Büchele          et al., 2003; Mathe et al, 2004).          Their retention time under the designed gradient          is shown in table 4. These triterpenes were assigned by a number,          according to their elution order. Number assignment will be kept all          along this paper for the identification of these molecules in          chromatograms.          Table 4: Retention time of standard          moleculesTableau 4 : Temps de rétention des          molécules standards                                                                          N°                                                                                      Molecule                                                                                      Retention time (min)                                                                                                              1                                                                                      3-                                                        epi                                                        -lupeol                                                                                      26.5                                                                                                              2                                                                                      lupeol                                                                                      27.6                                                                                                              3                                                                                      3-                                                        epi                                                        -                                                        β                                                        -amyrin                                                                                      28.2                                                                                                              4                                                                                      lupenone                                                                                      28.7                                                                                                              5                                                                                      3-                                                        epi                                                        -                                                        α                                                        -amyrin                                                                                      29.4                                                                                                              6                                                                                      β                                                        -amyrin                                                                                      29.9                                                                                                              7                                                                                      α                                                        -amyrin                                                                                      31.3                                                                                                              8                                                                                      β                                                        -amyrone                                                                                      31.4                                                                                                              9                                                                                      α                                                        -amyrone                                                                                      32.2                                                                                  Results of HPLC-UV/Vis study on certified          resins samples          The first difficulty for the study of resins by          HPLC-UV/Vis arose because of the need of different amounts of sample          depending on the apolar molecules content of the studied species:          going from 2 mg up to 30 mg, as referred to in table 5.          Table 5: Amount of resins used for the HPLC          chromatographic study and gum-resin percentage in resin samples from          certified species.Tableau 5 : Quantité de          résines employée pour l’analyse chromatographique et pourcentage en          gomme-résine des résines certifiées botaniquement.                                                                                        Mass of sample used for HPLC                    analysis                                                                                      % Gum                                                                                      % Resin                                                                                                              B. bipinnata                                                                                      3 mg                                                                                      0                                                                                      100                                                                                                              B. excelsa                                                                                      6 mg                                                                                      0.5                                                                                      99.5                                                                                                              B. simaruba                                                                                      30 mg                                                                                      25                                                                                      75                                                                                                              B. penicillata                                                                                      3 mg                                                                                      0                                                                                      100                                                                                                              B. stenophylla                                                                                      3 mg                                                                                      0                                                                                      100                                                                                                              B. laxiflora                                                                                      6 mg                                                                                      8                                                                                      92                                                                  The amount of resin needed for the analysis has          been directly correlated to the amount of gum present in the sample:          the higher amount of gum was, the rarer apolar compounds were          (terpenoids included) and so, the more important the sample amount          needed for the analysis          Table 5 also sums up the percentage of gum found          in each studied species. Generally gum ratio is correlated to the          producing species but this fact alone cannot constitute a factor for          botanical identification, as gums are water soluble, widely          distributed, and easy degradable materials. The important proportion          of gum in B. simaruba exudates provided          chromatograms with less resolved peaks than in other species,          therefore the results of analysis of this species could not be          treated by PCA and LDA statistical analysis.          Chromatograms in liquid phase allowed a very          clear interspecies distinction. It is note-worthy that two separated          areas were distinguished in each chromatogram: the first one going          from 0 to 24 minutes and the second running from 24 to 35 minutes.          The first zone corresponds to components with lower molecular weight          and highest polarity. This first molecular population was          characterized by a succinct LC-SM analysis as sesquiterpenes,          however these peaks cannot be surely assigned in the context of this          article. The second group of peaks in the chromatogram corresponds          to molecules of higher molecular weight and lower polarity; nine of          the eleven molecules detected in this region were identified by          co-injection with standard molecules as triterpenes.          Figure 1 shows a chromatogram of B. bipinnata where the two above mentioned areas          of peaks are distinguished: marked with a continuous arrow the more          polar zone, and with a dotted one the triterpenoid zone. For the          first part of the chromatogram we chose a notation for compounds          beginning either with letters A or B.          Compounds noted “A” are widely distributed in the          six species, most of the time A1 is a major peak in the          chromatograms. These peaks were present also in chromatograms of          archaeological samples. While “B” compounds are specific for each          species, meaning that their presence and quantity varies a lot among          studied species and were almost absent from archaeological          samples.          Figure 1: Chromatogram of B. bipinnata (sample 11d2), triterpenoid zone          running from 24 to 35 min in retention time. (1) 3-epi-lupeol, (2)          lupeol, (3) 3-epi- β amyrin, (4) lupenone, (5) 3-epi- α amyrin, (6)          β -amyrin, (7/8) α -amyrin/ β -amyrone, (9) α -amyrone).Figure 1 : C hromatogramme de B. bipinnata          (échantillon 11d2), agrandissement de la zone triterpénique de 24 à          35 min. (1) 3-épi-lupéol, (2) lupéol, (3) 3-épi- β amyrine, (4)          lupénone, (5) 3-épi- α amyrine, (6) β -amyrine, (7/8) α -amyrine/ β          -amyrone, (9) α -amyrone).                    Table 6 resumes the presence and absence of          compounds of lower molecular weight and higher polarity, and table 7          sums up the presence and absence of triterpenoids related to          botanical origin.          Table 6: Retention time of peak corresponding          to non triterpenic compounds observed in each species (+ presence, -          absence).Tableau 6 : Temps de rétention des          pics correspondants aux composés non triterpéniques de chaque résine          (+ présence, - absence).                                                                           ID of the peak                                                                                      A4                                                                                      A3                                                                                      A2                                                                                      A1                                                                                      B1                                                                                      B2                                                                                      B3                                                                                      B4                                                                                      B5                                                                                      B6                                                                                      B7                                                                                      B8                                                                                                              Ret. time (min)                                                                                      8.9                                                                                      10                                                                                      11.4                                                                                      12                                                                                      16                                                                                      16.5                                                                                      17                                                                                      18                                                                                      19.4                                                                                      20                                                                                      20.6                                                                                      24                                                                                                              B.bipinnata                                                                                      -                                                                                      +                                                                                      +                                                                                      +                                                                                      -                                                                                      -                                                                                      -                                                                                      +                                                                                      +                                                                                      -                                                                                      +                                                                                      -                                                                                                              B. stenophylla                                                                                      +                                                                                      +                                                                                      +                                                                                      +                                                                                      -                                                                                      -                                                                                      -                                                                                      +                                                                                      +                                                                                      -                                                                                      +                                                                                      +                                                                                                              B. excelsa                                                                                      -                                                                                      +                                                                                      -                                                                                      +                                                                                      +                                                                                      -                                                                                      -                                                                                      -                                                                                      +                                                                                      +                                                                                      -                                                                                      +                                                                        B. laxiflora(profile A)                                                    +                                                                                      +                                                                                      +                                                                                      +                                                                                      +                                                                                      +                                                                                      -                                                                                      -                                                                                      -                                                                                      -                                                                                      -                                                                                      +                                                                        B.laxiflora(profile B)                                                    -                                                                                      -                                                                                      +                                                                                      +                                                                                      +                                                                                      +                                                                                      -                                                                                      -                                                                                      -                                                                                      -                                                                                      -                                                                                      +                                                                                                              B. penicillata                                                                                      -                                                                                      -                                                                                      -                                                                                      +                                                                                      +                                                                                      -                                                                                      +                                                                                      +                                                                                      +                                                                                      -                                                                                      -                                                                                      -                                                                                                              B. simaruba                                                                                      -                                                                                      +                                                                                      +                                                                                      +                                                                                      -                                                                                      -                                                                                      -                                                                                      -                                                                                      -                                                                                      +                                                                                      -                                                                                      -                                                                                                              Archaeological sample A                                                                                      -                                                                                      +                                                                                      -                                                                                      +                                                                                      -                                                                                      -                                                                                      -                                                                                      +                                                                                      -                                                                                      -                                                                                      +                                                                                      +                                                                                                              Archaeological sample B                                                                                      +                                                                                      -                                                                                      -                                                                                      +                                                                                      -                                                                                      -                                                                                      -                                                                                      +                                                                                      -                                                                                      -                                                                                      -                                                                                      -                                                                  Table 7: Retention time of peaks          corresponding to triterpenic compounds (+ presence, - absence).          Tableau 7 : Temps de          rétention des pics des composés triterpéniques (+ présence, -          absence).                    Concerning resolution of peaks, in our analytical          conditions, it was impossible to separate the signals from α-amyrin          (7) and β-amyrone (8), which is not surprising as both compounds          have a similar molecular structure. The closest triterpenic          molecular composition was found between B.          bipinnata and B. stenophylla species. A          difference between B. bipinnata and B. stenophylla, lies in the absence of          triterpenic compounds T1 at 25.4 min and T2 at 33.5 min in B. bipinnata (Table 7, Figure 2). Gum was absent          of the composition of both species.          Figure 2- Comparison of A) B. bipinnata and B) B.          stenophylla chromatograms. (1) 3-epi-lupeol, (2) lupeol, (3) 3-epi- β -amyrin, (4) lupenone, (5) 3-epi- α amyrin, (6) β -amyrin, (7/8) α -amyrin/ β          -amyrone, (9) α –amyrone.Figure 2-          Comparaison des chromatogrammes de A) B. bipinnata et B) B.          stenophylla. (1) 3-épi-lupéol, (2) lupéol, (3) 3-épi- β -amyrine,          (4) lupénone, (5) 3-épi- α amyrine, (6) β -amyrine, (7/8) α          -amyrine/ β -amyrone, (9) α -amyrone.                    Concerning B. excelsa it          was the species that displayed the poorest composition in the          triterpenic zone, only four compounds were detected by HPLC-UV/Vis:          lupeol (2), 3-epi-lupeol (3), lupenone (4)          and β –amyrin (6). Nevertheless relative peak areas of β –amyrin (6)          and lupenone (4) were considerably smaller than the ones for those          of the other two compounds, and in the apolar zone A1 was the major          peak. On the other hand gum content of samples of this resin reaches          almost zero (0.5%).          B. simaruba resin          exhibited the higher content of gum (25%). The presence of gum          complicated HPLC analysis: resolution of peaks corresponding to          triterpenes in its chromatograms was poorer, three clumps of peaks          were observed, the first one consisting of 3-epi-lupeol, lupeol, 3-epi-β-amyrin and lupenone while the second one          3-epi-α amyrin and β –amyrin, the third          included the peak comprising 7/8 α-amyrin/ β –amyrone like the other          species plus α-amyrone, and in the more polar zone of the          chromatogram A1 was outnumbering the other peaks.          B. penicillata exhibited a          high number of peaks in the first zone of the chromatogram, where A1          was the highest, other peaks with high relative area were B2, B3 and          B5. Concerning the triterpenic zone, nine standard molecules were          identified; the only triterpenes absent from its composition were T1          and T2.          Finally B. laxiflora          resins were the ones that displayed the greater diversity in          molecular composition in this study. Among the samples analyzed, two          lots could be distinguished, given their diverse molecular profile.          Nevertheless some common features could be distinguished between the          two lots: B1 had the peak with the biggest area in all the cases.          Concerning triterpenic fraction, the relative peak area of          triterpenic compounds was rather small. In “lot a”, triterpenic most          important peaks were those from 3-epi-lupeol,          lupeol and 3-epi-β-amyrin, while in “lot b”          the main signal in the triterpenic zone was 3-epi-lupeol.                          Results of HPLC-UV/Vis study on          archaeological resins          In archaeological samples, the percentage of gum          was nul: this fact can be correlated either to the botanical origin          of the sample or to the fact that these samples were exposed during          a long time to high moisture and even under-water conditions.          Nevertheless sample B contained a small proportion (1.73) of matter          that was not soluble in our experimental conditions. The small          amount of this material available for analysis prevented further          studies on its nature.          Despite the age of archaeological samples and          their preservation under different conditions of relative humidity,          pH and temperature, their molecular composition (appearing in          chromatograms) retained most of the triterpenic fraction compounds          except for 3-epi- β-amyrin and T1, as it has          been said above. Within the more polar fraction the most important          signal arose from compound A1.          The exceptional conservation state of these          samples can be attributed to burial anoxic environment with a high          humidity level, the absence of light, and more or less stable          temperature.          HPLC chromatogram profiles from Aztec materials          coming from Templo Mayor may suggest that they are either from B. bipinnata or B.          stenophylla. In order to enhance the exploitation of these          results, and to find a more concrete answer regarding the botanical          origin of these samples, two statistical methods were used to          analyze the data.          Concerning the molecular profile , the          archaeological samples studied were different: in sample B four          molecules that were not present in fresh samples were detected, this          was the reason that made the use of a longer gradient necessary.          These compounds were absent from sample A.          In figure 3, the chromatogram corresponding to          sample B is displayed; the peaks can be appreciated from 32.5 to 36          min identified as C1 to C3. These compounds may correspond to apolar          molecules. In figure 4, the chromatogram of sample A is          presented.          Figure 3: Chromatogram of archeological          sample B with triterpenes and ageing related products (C1-C3). 1)          3-epi-lupeol, 2) lupeol, 3) 3-epi- β -amyrin, 4) lupenone, 5) 3-epi- α amyrin, 6) β -amyrin, 7/8 α -amyrin/ β          -amyrone.          Figure 3 : Chromatogramme          de l’échantillon archéologique B avec la présence de triterpènes et          de composés de dégradation (C1-C3). 1) 3-épilupeol, 2) lupéol, 3)          3-épi- β -amyrine, 4) lupénone, 5) 3-épi- α amyrine, 6) β -amyrine,          7/8 α -amyrine/ β -amyrone.                    Figure 4: Chromatogram from archeological          sample A. 1) 3-epi-lupeol, 2) lupeol, 3)          3-epi- β -amyrin, 4) lupenone, 5) 3-epi- α amyrin, 6) β -amyrin, 7/8 α -amyrin/ β          -amyrone, 9) α -amyrone.Figure 4 :          Chromatogramme de l’échantillon archéologique A.1) 3-épi-lupeol, 2)          lupéol, 3) 3-épi- β -amyrin, 4) lupénone, 5) 3-épi- α amyrine, 6) β          -amyrine, 7/8 α -amyrine/ β -amyrone, 9) α -amyrone.                    From these chromatograms, important informations          can be extracted concerning ageing: some molecules disappeared in          some cases like A2, A3, B8 Molecules that were totally absent from          these samples and that therefore are more prone to degradation are:          B1, B2, B3 and B5.          Finally sometimes molecules related to ageing          like C1 to C3 may appear. However some compounds resist to ageing          like triterpenes (1-8).                          HPLC coupled to PCA analysis for botanical          certified resins          For this study, both polar (table 6) and          triterpenic compounds (table 7) were taken into account. Peak areas          from chromatograms were used in an indirect way: as HPLC analyses          were performed on different amounts of crude resin, we chose to use          the relative area percentage of each peak for PCA and LDA          analysis.          B. simaruba was excluded          from statistical analysis because of the poor resolution achieved          that prevented distinction of the peaks of triterpenic compounds. A          data matrix with 40 rows (resin of certified origin samples) and 22          columns (variables, in this case relative area percentage of the          assigned peaks in the chromatogram) was built. Figure 5 shows the          graphic results of PCA. The structure of the data, using the first          two principal components is displayed.          In this case the first two principal components          were enough to display the data structure, and they explained 46.5%          of the total variance. By examining the score plot in the area          defined by the first two principal components, good discrimination          results were achieved, furthermore even B.          bipinnata and B. stenophylla were          correctly discriminated. B. penicillata is          also well separated from the other species.          Concerning B. excelsa and B. laxiflora          they were grouped into the same area of hyperspace. Both species          have a lower content of triterpenes compared to the other studied          species, and this factor leads to a loss of sensibility in their          discrimination using PCA with HPLC data (Figure 5).          Figure 5: Distribution PCA of the resins          samples from botanical certified origin with HPLC data. Figure 5 : Représentation ACP des résines certifiées          botaniquement, en les données CLHP.                    ● B. laxiflora, ◆B. excelsa, ■ B.          stenophylla, ▲ B. bipinnata, ▼ B. penicillata          When analyzing the loading plot (Figure 6), the          molecules that exhibit the highest influence onto first component          with positive values are peaks corresponding to triterpenes:          lupenone, 3-epi-α-amyrin, lupenone, α-amyrin/          β-amyrone. On the negative side with the highest impact B8 and B1          from the more polar fraction have a highest discrimination          power.          On the negative part of first component B2, B5,          A3 and A4, all volatile compounds have been shown to have a higher          impact, together with 3-epi-lupeol from          triterpenic fraction.          On the second component B3 and B5 have the higher          impact in discrimination, they both seem to own the same information          so for further analysis B5 may be taken out of the analysis as it          exhibits a slightly lower impact on both components. Along with B3          and B5, α-amyrone was found to have a high impact on second          component. On the negative part of the second component A3, A4 and          3-epi-lupeol display the higher          discrimination power.          Generally all triterpenes play a major role into          discrimination except for lupeol which seems to have little impact          on both components, and A1 which also exhibits a very limited          influence into discrimination. This is reasonable if its ubiquity is          considered.          Figure 6: Loading plot for PCA analysis using          HPLC data for botanical certified resins.Figure 6 : Graphique des variables en ACP utilisant          les données HPLC des résines certifiées botaniquement.                                    Linear Discriminant Analysis          A classification model was built based on the          same matrix used for PCA. LDA analysis was applied in order to find          a predictive classification model, able to separate the five          botanical species. Results for fitting analysis are shown on table 8          and in table 9for validation matrix.          Table 8: Fitting matrix of the LDA Rows          represent the true class, and the columns the assigned          class.Tableau 8 : Matrices d’ajustage en          analyse linéaire discriminante, les lignes représentent la vraie          classe d’appartenance, et les colonnes les classes          attribuées.                                                                          Put into group                                                                                      B. bipinnata                                                                                      B. excelsa                                                                                      B. laxiflora                                                                                      B. penicillata                                                                                      B. stenophylla                                                                                                              B. bipinnata                                                                                      10                                                                                      0                                                                                      0                                                                                      0                                                                                      0                                                                                                              B. excelsa                                                                                      0                                                                                      4                                                                                      0                                                                                      0                                                                                      0                                                                                                              B. laxiflora                                                                                      0                                                                                      0                                                                                      12                                                                                      0                                                                                      0                                                                                                              B. penicillata                                                                                      0                                                                                      0                                                                                      0                                                                                      6                                                                                      0                                                                                                              B. stenophylla                                                                                      0                                                                                      0                                                                                      0                                                                                      0                                                                                      8                                                                                                              Total N                                                                                      10                                                                                      4                                                                                      12                                                                                      6                                                                                      8                                                                                                              N correct                                                                                      10                                                                                      4                                                                                      12                                                                                      6                                                                                      8                                                                                                              Proportion                                                                                      1                                                                                      1                                                                                      1                                                                                      1                                                                                      1                                                                                                              N = 40                                                                                      N Correct = 40                                                                                      Correct Proportion = 100%                                                                  The confusion matrix (table 9) allowed a global          recognition percentage of 95.0% while only one sample of B. excelsa and one from B.          stenophylla samples were incorrectly classified in the          validation procedure.          This can be considered as a consequence of          dealing with classification from a sample of B.          stenophylla, so that their probability to be correctly          classified by this model is 87.5 %. On the other side B. bipinnata was always correctly classified. When looking at figure 5 with PCA          classification, a greater dispersion of the individuals          corresponding to B. stenophylla can be          noticed, this fact leading to a greater possibility of          misclassification of resin from this origin, when applying this          model with the present data.          Table 9: Confusion matrix of LDA.Tableau 9 : Matrices de confusion en ADL.                                                                          Put into group                                                                                      B. bipinnata                                                                                      B. excelsa                                                                                      B. laxiflora                                                                                      B. penicillata                                                                                      B. stenophylla                                                                                                              B. bipinnata                                                                                      10                                                                                      0                                                                                      0                                                                                      0                                                                                      1*                                                                                                              B. excelsa                                                                                      0                                                                                      3                                                                                      0                                                                                      0                                                                                      0                                                                                                              B. laxiflora                                                                                      0                                                                                      1*                                                                                      12                                                                                      0                                                                                      0                                                                                                              B. penicillata                                                                                      0                                                                                      0                                                                                      0                                                                                      6                                                                                      0                                                                                                              B. stenophylla                                                                                      0                                                                                      0                                                                                      0                                                                                      0                                                                                      7                                                                                                              Total N                                                                                      10                                                                                      4                                                                                      12                                                                                      6                                                                                      8                                                                                                              N correct                                                                                      10                                                                                      3                                                                                      12                                                                                      6                                                                                      7                                                                                                              Proportion                                                                                      1                                                                                      0.75                                                                                      1                                                                                      1                                                                                      0.875                                                                                                              N = 40                                                                                      N Correct = 38                                                                                      Correct Proportion = 95%                                                                  Rows represent the true class, and the          columns the assigned class. Wrong assignments are marked with a          *.Les lignes représentent la vraie classe et          les colonnes la classe attribuée. Les attributions incorrectes sont          marqués d’une *.                          HPLC coupled to PCA analysis for          archaeological resins          Data concerning the two archaeological samples          from Templo Mayor were added into the matrix in an attempt to          achieve the discrimination of their botanical origin. In this graph,          it could only confirm the previous interpretation based only on          chromatograms in which an origin between B.          bipinnata and B. stenophylla is          suggested (fig. 4). Given the closeness of these two species in the          hyperspace it was not possible to assess with certainty the          botanical origin of the archaeological samples. This fact is also          connected to the molecular changes that take place during ageing          processes.          Figure 7: Distribution PCA of the resins          samples from botanical certified origin including archaeological          samples with HPLC data.Figure 7 :          Représentation ACP des résines certifiées botaniquement et des          échantillons archéologiques.          ●B. laxiflora, ♦ B. excelsa, ■ B. stenophylla, ▲ B.          bipinnata, ▼ B. penicillata, ● A, ● B                      4. Conclusion - Discussion         The usefulness of HPLC-UV/Vis was undeniable        concerning molecular profiling for differentiation between resins with        different botanical origin. Currently the botanical distinction        between B. stenophylla and B.        bipinnata is unclear and the use of very techniques like        HPLC-UV/Vis allows obtaining slight differences in the molecular        composition of their resins. This closeness on molecular composition        confirms the genetic closeness of these two species.        The advantage of coupling this liquid        chromatographic technique with statistical methods such as PCA and LDA        was to obtain a model able to discriminate botanical origin of fresh        resins from species with similar molecular compositions such as B. bipinnata and B.        stenophylla, which were difficult to differentiate using only        chromatographic results. The performance of the mathematical model        constructed with HPLC data in LDA shows a high resultwith 100%        recognition in the fitting matrix and a 95% of positive recognition in        the cross validation matrix.        Concerning ageing, liquid chromatography confirms        that Bursera resins undergo significant changes        over time: on the one hand a decrease of the polar compounds        composition and on the other hand an oxidation and maybe a        polymerization of the triterpenic fraction. These modifications can be        assessed only partially by means of HPLC-UV/Vis, as it was presented        on the chromatograms from archaeological samples and therefore a        deepened study should imply the isolation of compounds C1 to C3 and        its formal study by N.M.R. or MS-MS. Therefore there is a problem of        loss concerning some compounds and the polymerization of others that        lead to sometimes drastic changes in proportions between the        compounds. It is also thought out to make an artificial ageing study        of contemporary resins.        It is interesting to notice that some compounds are        not always present in archaeological resins. Therefore an insight into        the mechanism of their formation and maybe a correlation between        ageing conditions and their presence should be established.        In order to achieve a better botanical        discrimination in aged samples, further studies need to be conducted        about degradation processes, including the identification of        non-triterpenic molecules and also molecules arisen from ageing.        In a second step, the establishment of correlations        between products of degradation and stages of ageing could be        established. Only a few molecules seem to be stable during natural        ageing: A1 of the non-triterpenic fraction and all the triterpenic        molecules except 3-epi-β-amyrin, which        nevertheless exhibited a tendency to proportion change.        Molecular profile of both archaeological resins is        quite close despite a slight difference in conservation state.        Generally speaking, burial environment contributes to a extent to the        good preservation of these materials based on molecular profile for        both samples analyzed in this work, even though they were collected        from different offerings (offerings 120 and 126). The analysis results        on archaeological samples suggest that they could be resins of either        B. bipinnata or B.        stenophylla species. Pollen of B.        bipinnata has been founded by paleobotanists, in archaeological        context in the site of Templo Mayor (Montufar, 2007). However, B. bipinnata is a species with a wide geographical        presence and sometimes it produces hybrids with other members of the        Bullockia botanical section.        On the historical part, the results of these        experiments confirmed the accuracy of information contained in        chronicles from 16th century from B. Sahagún about the numerous uses        that Aztecs and original people from Mesoamerica gave to copal. This        research also yielded important information for archaeologist that was        previously unavailable: firstly this work confirms, on the molecular        level, that materials found at Templo Mayor offerings are natural        solid resins, and that one botanical origin of copal among the great        variety of over 80 possible species of Bursera        available at Aztec empire, seems to be preferred by the        priests.