Paola Lucero
email: paola.lucero@etd.univ-avignon.fr

Paola

CHAPTER I GENERAL PRESENTATION OF THE COPALS

Keywords: Aztec, Maya, Mexican copal, triterpenoids, archeological chemistry, archeological figurine, aztec adhesive, plant resin, HPLC-UV/VIS, FTIR, GC-MS, PCA, LDA, Bursera spp

4 1.1 General presentation of Mexican copals 1.2 Botanical and geographical distribution 1.2.1 Botanical sources and collection 1.3 Historical Context 1.3.1 The Maya civilization 1.3.2 Chichén Itzá city 1.3.3 Importance of "Sacred Cenote" at Chichén Itzá and their offerings 1.4 The Aztec civilization 1.4.1 The foundation of Mexico-Tenochtitlán 1.4.2 The rise of Aztec empire 1.4.3 The importance of "Templo Mayor" site 1.4.4 The building stages in Templo Mayor construction 1.5 Uses and properties 1.5.1 Copal uses in ancient Mexico 1.5.2 Contemporary uses and applications of copal 1.6 Previous works on chemical composition of Mexican copals 1.7 Oleo-gum-resin: definitions 1.8 Gums 1.9 Gum composition of Bursera simaruba 1.10 Fats 1.11 Terpenic resins 1.12 Bio synthesis of terpenic precursors 1.13 The essential oils 1.13.1 Triterpens 1.13.2 The ursans 1.13.3 The oleanans 1.13.4 The lupans 1.13.5 Biological activity

CHAPTER II PRESENTATION OF THE COPAL SAMPLES AND THE ANALYTICAL STRATEGY

Extraction of copal

This thesis would not have been possible without the help and advice of many scientists, restorers, and archaeologist.

It is my deep wish to thank all these people:

First of all, I wish to thank Prof. Dr. Cathy Vieillescazes for giving me the opportunity to work in her group on this exceptional research project. She supported me whenever necessary and made this thesis a great learning experience.

My most genuine gratitude is owed to Dr. Carole Mathe I thank her for her guidance. Her support and knowledge in conservation science was very valuable and helpful.

Many special thanks go to Prof Dr. Irma Belio and Prof Dr. Lauro Bucio. They contributed considerably to the success of this work with their brilliant ideas, on statistical and mathematical treatment of data. I thank them as well for their active collaboration in the collection of certified samples. I cordially thank them for his friendship and enthusiasm and unconditional support.

I am grateful to Dr. Rito Vega, without his precious knowledge in botanic, collection of certified samples would not have been possible. I thank Dr. Leonardo López Luján director of the archaeological project of Templo Mayor, for granting us access to both the archaeological site and the archaeological samples, and for the confidence that he deposed on this scientific work and its results.

It was a great experience to talk, with María Barajas and the members of the team of the Conservation Department of Templo Mayor Museum. I really appreciate the discussions with them, because they taught me what the problems in restoration are, and which improvements and knowledge would be helpful.

I also wish to thank Dr. Aurora Montúfar, who AKNOWLEDGMENTS I cordially thank the colleagues from the electronic microscop at the Physiscs Institute from the UNAM fro their collaboration in the analysis of copal samples.

Most grateful I am to Niko for his support, patience and for following me in this project.

A very special thank goes to all my friends that always support me, I will always remember you.

Finally my love and gratitude goes to my family that helped me and guided me all along my studies.

PART B Analytical Study of the resins and Discussion 57 CHAPTER III SPECTROSCOPIC TECHNIQUES: FTIR

3.1 Mathematical Treatment of Experimental data: chemometric strategies 3.1.1 Principal Component Analysis 3.1.2 Linear discriminant analysis (LDA) 3.2 Chemotaxonomy. Correlation of resin composition and botanical origin 3.2.1 Terpenoids and chemotaxonomy 3.3 Study by Fourier-transformed infrared spectroscopy (FTIR) 3.3.1 FTIR spectra of terpene and terpene like molecules 3.4 Sampling Methods: transmission methods 3.5 Reflectance Methods 3.6 Results and discussion 3.7 PCA 3.8 Linear discriminant analysis 3.9 Application to a botanical origin certified sample 3.10 Application to two commercial samples of copal 3.11 Deconvolution of the bands in the region between 1200 and 1300 cm -1 of the spectra. Application of the technique to two commercial resins 3.12 Application to archeological resins 3.13 Partial analysis of the surface of the samples 3.13.1 Comparison of partial analysis from commercial samples and a naturally aged sample of them.

V Table 1 Monosaccharide composition of B. Simaruba [START_REF] Magaloni | Los pintores de Bonampak[END_REF] Chapter II Presentation of the Analytical Strategy and the copal samples Table 2 Copals collected in 2004-2005 Table 3 Copals collected in 2008 Table 4 Copals collected in 2010 Table 5 Arqueological Aztec samples Table 6 Archeological sample from Chichén Itzá

Liste of Figures

PART B Analytical Study of the resins and Discussion

Chapter III Spectroscopic Techniques: FTIR

Table 7

Assignment for the most characteristic IR bands of some terpenoid compounds [START_REF] Schulz | Identification and quantification of valuable plant substances by infrared and Raman spectroscopy[END_REF] Table 8 Band positions taken into account as variables for the PCA matrix Table 9 Results for LDA classification model: Fitting and validation matrix were identical. Rows represent the true class, and columns the assigned class. Wrong assignments are shown in red

Chapter IV High Performance Liquid Chromatography -UV/Vis Analysis 16 Retention time of peak corresponding to non triterpenic compounds founded in each species (+ presence, -absence)

96

Table 17 Retention time of peaks corresponding to triterpenic compounds found in each species (+ presence, -absence)

96

Table 18 Fitting matrix of the LDA Rows represent the true class, and the columns the assigned class LDA results with HPLC data for certified samples: fitting and confusion matrix

Table 47

Concentration of standard molecules in botanically certified samples (mg/ ml)

Table 48

Concentration of standard molecules in archaeological samples (mg/ ml)

Table 49

Concentration of lupenone in B. simaruba samples (mg/ ml) Table 50 Concentration of standard molecules in commercial samples (mg/ ml) Table 51 Retention times of standard molecules under gradient 2 for the study of triterpenic compounds

Table 52

Retention times of standard molecules under gradient 1 for the study of archaeological samples

The Maya civilization

Yucatan peninsula climate is characterized by recurring hurricanes, savannas pound to flooding, a cave system that drain rainwater, a lack of surface groundwater, and a 6 to 7 months of dry season [START_REF] Faust | Mayan environemental succeses and failures in the Yucatan Peninsula[END_REF].

Maya culture is one of the most important and sophisticated ancient cultures, from the Mesoamerican cultural area of pre-Columbian time [START_REF] Vandenabeele | Raman spectroscopic analysis of the Maya wall paintings in Ek'Balam, Mexico[END_REF]. The first settlements of Maya civilization date, from 3500 B.C., in the Yucatan peninsula and, continued until 200 A.D. in a phase known as the Preclassic period. Considering Yucatán environment, water provision was a major concern to the Maya and that is why during the Preclassic phase cities as Edzná and Tikal were constructed with extensive systems for collecting water, using reservoirs and underground tanks. While other cities as Ek Balám, Mayapán and Loltún constructed basins and ditching channels for the management of water (Andrews et al, 1960). In the next map (figure 5) the southern classical sites are showed and the northern post classical sites, including Chichén Itzá. But undoubtedly the most remarkable building of all the city is the Kukulcán pyramid (Mayan equivalent of Quetzalcóatl God), also known as "the Castle". In this building twice a year: in the spring equinox and in autumn solstice a game of shades on the building simulates a serpent that descends until loosing itself in the Earth.

The Castle as other important pyramids in Mesoamerica (like the Templo Mayor) hides an older pyramid inside itself. In its interior there are a Chac Mool (God associated to the rain cult) and a throne of jaguar painted in red with incrustations of jade simulating their skin. This building has been interpreted like the material expression of a calendar. In fact if the number of steps of the four sides and those of the entrance of the temple are added, they sum 365, the same number than the days of the year.

Importance of "Sacred Cenote" at Chichén Itzá and their offerings

Cenotes are natural sinkholes in the limestone of rock that forms the geological base of the peninsule. Among Maya, cenotes were considered as sacred places, and offerings were common.

Chichén Itzá is a Classic site, established on the Northern lowlands of Yucatán (figure 6), "In the border of the well of the Itzá" (Andrews, 1990), undoubtedly a reference to the sacrifice cenote.

The city had a height towards years 600 and 900 B.C. soon to be lefted and to be refunded by the "itzáes" in 500 A.D. It was the Mayan priest Lakin Chan, known also as Itzamná that brought new blossoming to this large city. From that moment, their inhabitants named themselves chanes or itzáes. The Chichén Itzá complex counts with two cenotes: the "Xtoloc" in the west side, and the Sacred or Sacrificial on the east side.

Since the first explorations of Chichén Itzá, copal was recovered from sacred Cenote. It is no surprising as it was a worship place, where not only Chichén Itza inhabitants celebrated important ceremonies, but also a peregrination site where Mayas from distant places deposed offerings as well [START_REF] Morley | La civilización Maya[END_REF].

Between 1904 and 1911, Edward H. Thompson explored and, drained Sacred Cenote, finding over 100 gold objects, bones, ceramic vessels, shells, a scepter with anthropomorphic form, 250 objects of jade, pectorals, lances of flint, textiles of cotton, pumpkins but according to their notes "the most abundant material is some pom-incense pellets, often found into the bottom of blue painted vessels, where copal was "molded or poured before being deposed into the Cenote" [START_REF] Coggings | Artifacts from the Cenote of Sacrifice, Chichen Itza, Yucatan: textiles, basketry, stone, bone, shell, ceramics, wood, copal, rubber, other organic materials, and mammalian remains[END_REF].

Maya preparation of copal, was quite complex, the copal was worked until it became malleable, copal soot mixed with "Maya blue", was applied to the surface of ceramic pieces. In other occasions, different tree exudates were applied to the surface of the object to offer, like chicle (from Manilkara zapota) or rubber (from Castilla elastica). The pattern of the copal varied with round, rolled pellets, bars in a conglomerate and cones.

In the archaeobotanical collection of the INAH (National Institute of Anthropology and History of Mexico) over 200 pieces of copal extracted from the Sacred Cenote of Chichén Itza are preserved. Among these pieces there is one ball molded with ulli (rubber) incrustations, another ball with corn grained applications on its surface, and a human face (Montufar, 2007).

The Aztec civilization

The Mexicas or Aztecs were an ethnic group with nahua filiation, that after a long peregrination funded México-Tenochtitlan, a city that between the XV th and XVI th centuries became the center of one of the biggest empires of all the times in Mesoamerica. Aztecs developed rich and complex religious, political, cosmologic, astronomic, philosophic and artistic traditions. In a good amount Aztecs were the heirs of the cultures of many other ancient Mesoamerican civilizations as the Olmecs, the Teotihuacans and the Toltecs.

The rise of Aztec empire

The importance of this alliance relies on the fact that in barely 100 years, it conquered a big territory: from Atlantic to Pacific Oceans of current territory of Mexico. After a couple of years Aztecs took control over their allies, dominating them thus giving rise to the Aztec empire (Obregon-Rodriguez, 2001).

This empire, as many other empires in history, excerpted an effective political, economic and ideological control over the dominated territory, Aztecs got a big city, as capital of the empire:

Mexico-Tenochtitlan, where the proclamations of imperial ideology where marked and reflected in the architecture of the buildings [START_REF] Smith | The archeological study of empires and imperialism in prehispanic central Mexico[END_REF]. At the heart of this imperial ideology we find the "Templo Mayor".

The importance of "Templo Mayor" site

The "Templo Mayor" was built at the geographical center of the city it was a precinct surrounded by a staggered platform, where 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.

At this temple took place all ritual celebrations: from the regular ones, marked in pre Columbian calendar to the most extraordinary ones corresponding to the enthronization and funerary ceremonies for the emperor (Lopez-Luján, 2010). In each of these events offerings were buried [START_REF] Matos-Moctezuma | De conservaciones y restauraciones[END_REF].

The building stages in Templo Mayor construction

Although the position of Templo Mayor was known long time ago, it was believed to had been totally destroyed. Since at the moment of the defeat of the Aztecs, all the buildings of the city were destroyed and its materials employed in the construction of European style constructions. It was until 1978 that some excavations of the electricity company found the Coyolxhauqui (Moon Goddess) sculpture. This sculpture is important as documentary sources established its location at the foot of the stairs of Huitzilopoztli altar (Lopez Austin, 2001).

From this moment a program of excavation, conservation and preservation was designed [START_REF] Renfrew | El proyecto Templo Mayor[END_REF]. This archeological work, has shown that the construction of this ceremonial center was divided in 7 stages, corresponding more or less with each emperor reign, meaning that each ruler addressed a campaign of enlargement of the temple, that consisted in the construction of a superimposed pyramid onto the previous one, preserving the east-west orientation of the building (figure 7)

By the time the Spanish arrived to Tenochtitlan, Templo Mayor reached 60 m height (Matos- Moctezuma, 1987). It was a double Temple, the northern part was dedicated to Tlaloc (God of the rain), while the southern part was dedicated to Huitzilopochtli (God of War).

The Templo Mayor precinct measured 500 m on each side, having a total surface of 250 thousand square meters.

18

The first stage was probably made of wood, and to our days it is impossible to know if in the inside the remains of the pyramid there are still some remains of the first stage.

The second stage was constructed before 1428 A.D. and is visible now on, Mexico city downtown. There are some paintings preserved on the Tlaloc adoratory as well as a Chac-Mool.

In the last staircase just before a sacrificial stone there is a glyph that identifies this stage with a date that in our calendar would correspond to 1390 A.D. This is the earliest fully exposed structure, so its orientation, dimensions and structural characteristics have been measured and described [START_REF] Aveni | Myth, environment and the orientation of the Templo Mayor of Tenochtitlan[END_REF].

The third stage dates from 1431 A.D. (year 4 cane in pre Columbian calendar) that corresponds to Itzcoatl reign. From this period only some stairs and part of the paved are preserved.

The fourth stage present two constructive periods: the first one (dating from around 1454 A.D.) corresponds to Moctezuma I government. From this period, were recovered a good number of ornamental elements such as braziers dedicated to the regent gods of the temple, and the Coyolxauqhi sculpture.

The second constructive period corresponds maybe to the Axayacatl reign, where a platform ornamented with undulating serpents, was added to the front of the pyramid.

From the fifth constructive stage, remain few elements: a stuccoed platform and, part of the ceremonial floor dated from 1470 A.D.

From the sixth stage survived a fragment of the main facade with a wall that shows as ornamentation a sculpture of the head of three serpents. This stage dates from 1500 A.D.

Finally from the seventh 7 stage, which is dated between 1500 and 1521 A.D., remain the floor and the stairs that gave access to the Templo Mayor.

After and during the Spanish conquest of Mexico, all the buildings of the city were partially or totally destroyed, their remains as it was said before, were used as quarry for the edification of European buildings, and their foundations some times were re-used for these new constructions.

1.5

Uses and properties

Copal uses in ancient Mexico

As it has been said before on section 1.1 of this work, the first Spanish in American continent retained the term mexican term "copal" as well as the term "incienso de indias" (incense from Mexicans use copal quite often, at least twice a day, one in the morning and one in the afternoon.

It was used as an offering to the gods, to "clean" the holy places, during funerals, in offerings to ask for rains, etc. There was no important ceremony that took place without it. The representation of copal can often be found in codex where priest are offering it to the gods, or are keeping it in a special bag (figure 8).

In addition copal was used as a binder mixed with pigments, for the decoration of murals. In goldsmith, copal was used as the wax is used in our days, in the "lost wax" technique. It was part of the formulations that were used as adhesives for masks, small rocks, and even in dentistry (figure 9) where it was used in dental incrustations in teeth cavities and for dental restorations as well [START_REF] Bucio | Incrustation of precious stones in dental apatite[END_REF]. According to Orta Amaro, 2007, there are evidences that Mesoamerican cultures, from X th to VI th centuries A.D. used both copal and calcium phosphate-based cement to fix precious stones to perform dental incrustations, some reports in the literature indicate that pyrite, hematite, jadeite and turquoise were typically used for this purpose. The technique probably consisted in creating a cavity on the tooth surface (of frontal teeth) by means of a drill like tool and perhaps also using PAOLA LUCERO 20 powdered quartz with water as an abrasive. The stone was then fixed to obtain the incrustation in the dental tissue [START_REF] Orta-Amaro | Copal: Microestructura, composicion y algunas propiedades relevantes[END_REF].

Scale 1 cm picture: 2cm 1 cm picture: 10cm 1 cm picture: 1cm 1 cm picture: 12cm on their faces and as corporal paint for their foot". They painted themselves with red, yellow and dark preparations that were obtained mixing the resin with colorants; these mixtures were sometimes heated to obtain different gradation in colors (Anaya, 1998).

In traditional medicine copal has been reported to be used in cataplasm, for the treatment of skin infections [START_REF] Campos-Osorno | Proceso de Exrtacción de copal en una comunidad de la Sierra de Huautla[END_REF].

Miguel Venegas, a jesuit misioner, from XVIII th century, wrote in his book "Natural and civil history of California" that in San Bernabé bay, California, "the abundance of copal and incense trees allows to use their resin mixed with grease for waterproofing wooden ships, instead of pitch" (Venegas, 1757).

In the other hand, the gum produced by B. simaruba has been established to enter in the composition of the mural paintings binders from the Maya site of Bonampak [START_REF] Magaloni | Los pintores de Bonampak[END_REF].

Contemporary uses and applications of copal

Currently, the ancient curative and adivinatory ritual uses of copal are maintained in some Mexican villages [START_REF] Viveros | Interest ceremonial plants in two indigenous communities of the centromontaña region of Guerrero State, Mexico. A territorial approach[END_REF]. Copal can be easily found in several seasonal and permanent markets that will be furthered described in the second part of this chapter.

In traditional contemporary markets, copal is sold and recommended in infusion, as a cure for bronchitis. As a cataplasm, it is used to relief rheumatic pain. For the treatment of skin infections

21

and ulcers, traditional medicine recommends to heat copal until melting and to apply it, directly into the skin (Caralampio, 2000).

A few years ago, before the industrialization, along with Manilkara zapota resin, copal was used as chewing gum, that prevented cavities, and other dental affections, due to its chemical composition.

In the first days of November the "day of the dead" is still celebrated everywhere through the center and southern regions of actual Mexican territory. These celebrations include the elaboration of altars for the dead ancestors, that always include copal. In a notable synthesis,

where Christian and Native American rites are mixed [START_REF] Purata | Uso y manejo de los copales aromáticos: resinas y aceites[END_REF].

In the industry it is known to be part of the formulation of paintings, adhesives, artificial fireworks, soap, inks, varnishes and cosmetics creams (Montufar, 2007).

The essential oils are used widely like additives in food, fragrances, aromatherapy and traditional and alternative medicine. Synthetic and natural terpenoids have expanded the variety of used aromas.

In the patrimony field a binder made on copal has been used by outstanding painters such as

Gerardo Murillo (1875-1964), Diego Rivera (1886-1957), and David Alfaro Siqueiros (1896[START_REF] Cortes | Pegamentos, Gomas y Resinas en el México Prehispánico (Sticky-Materials, Gums and Resins in Prehispanic Mexico)[END_REF]. Rivera specifically used this type of resin in the mural «La creación». In this case he used a mixture of Mexican copal, elemi resin and wax [START_REF] López | Aplicaciones del copal mexicano como aglutinante pictórico[END_REF].

Previous works on chemical composition of Mexican copals

To our knowledge, there are few scientific studies that deal with chemical composition of Nevertheless, up to now none of these studies had studied resins from certified botanic origin.

The importance of the present work relies on the fact that our team, in collaboration with specialists in botanic from the Autonomous University of Sinaloa as well as the National University of Mexico, collected in situ the resins from certified botanical trees.

The important disadvantage of using commercial samples is that in many cases, as our colleague Irma Belio referred (personal communication) "the artisans collect resins, using the same 2000).

1.7

Oleo-gum-resin: definitions

Tree exudates are chemically complex mixtures of fluid terpenoids, with a variable ratio of volatile and non volatile components depending on their botanical origin. Triterpenoids are the primary constituents of resins in Burseraceae that produce a balsamic type terpenoid resin, while diterpenoids are the primary components in non volatile fraction from Conifers and most Angiosperms. Finally the gum fraction is composed by polysaccharides.

Gums

Chemically gums are formed by complex chains of hydrophilic polysaccharides (complex sugars) derived from simple sugars such as galactose, arabinose, rhamnose. In 1993 Whistler described in detail, the complex structure of gum exudates [START_REF] Langenheim | Plant resin, Chemistry, evolution, ecology, ethnobotany[END_REF]. The Burseraceae family also yields several resins, which may contain gum in some extent (De la Cruz-Cañizares, 2005).

Gum composition of Bursera simaruba

Among the gums produced by Mexican Bursera species, only the gum of B. simaruba has been studied [START_REF] Magaloni | Los pintores de Bonampak[END_REF]. Using an hydrolysis treatment before a GC-MS analysis, she found its monosaccharide composition (Table 1).

Peak relative intensity

Arabinose 38

Ramnose 4

Galactose 100

Glucose 3

Fats

Fats are chemically constituted by fatty acids. In the synthesis of fatty acids from carbohydrates, that is followed by the enzymatic combination of the carbohydrates with glycerol some esters may be formed. Plant fats differ from oils in having more or less solid fatty acid constituents, at normal temperature. Fatty acids occur in some Conifer resins (Langenheim, 2005). A study conducted by Van Gemert and Armitage in 2007 identified by means of GC-MS the next fatty acids in commercial copals: capric, decanoic, palmitic, tetradecanoic, caproic, propanoic and nonanoic acids. Nevertheless because of the uncertainty on botanical origin it is not possible to know if these acids were present originally in the resin or are due to pollution of the samples.

Terpenic resins

Resins are chemically constituted by terpens. They are a diverse family of compounds present in all living organism but they attain their greatest structural and functional diversity in plants, [START_REF] Langenheim | Plant resin, Chemistry, evolution, ecology, ethnobotany[END_REF] particularly Conifers, and Buseraceae. The name terpen is derived from the word turpentin (turpentine spirit). When the terpens are chemically modified by oxidation, the resulting compounds are referred generally as "terpenoids", some authors use invariably both terms.

This family includes the monoterpenes, sesquiterpenes, diterpenes, steroids, carotenes, polyprenoids such as ubiquinone side chain, and the polymer rubber. Although, their variability they are all synthesized by two pathways that will be further discussed.

Bio synthesis of terpenic precursors

The basic biological isoprenoid unit is isopentenyl diphosphate (IPP). For many years it was assumed that the early stages of the terpenoid biosynthetic pathway were the same for all types of terpenoids. This is now known to be untrue. In plants, IPP can be synthesized in either the cytosol or in the stroma of plastids, using different precursors at each site. The cytosolic pathway utilizes acetate and is named the mevalonate (MVA) pathway after the important intermediate, mevalonic acid. This is the only pathway that operates for terpenoid biosynthesis in fungi and animals.

The plastidic pathway in the green parts of plants and in algae uses glyceraldehyde 3-phosphate and pyruvate, and again the pathway is named for an important intermediate, methyl erythritol phosphate (MEP) (Kashara et al, 2002). A synthetic overview of the two pathways is shown on

1.13

The essential oils Some components of resins are referred as "essential oils" (Mills and White, 1977). In the case of Burseraceae resins, these oils are chemically mainly mono and sesquiterpens. The concentration of monoterpens in natural fresh resins always dominates over that of sesquiterpens and triterpenes. Monoterpens and sesquiterpens play a major defense role against insects and pathogens [START_REF] Langenheim | Plant resin, Chemistry, evolution, ecology, ethnobotany[END_REF].

Triterpenes

Concerning the "heavy" fraction of Burserseraceae resins, triterpenes with tetra or pentacyclic skeletons characterize them (figure 12). Mexican trees ursan compounds have been identified (figure 14). Cañizares, 2005) or archeological mexican resins [START_REF] Stacey | Chemical Characterization of Ancient Mesoamerican Copal Resins: Preliminary Results[END_REF]. Their structures are shown below, figure 16.

R 1 R 2 R 3 R 4 -OH -H -H -OH CHAPTER I GENERAL PRESENTATION OF COPALS PAOLA LUCERO 29

Biological activity

Pentacyclic triterpenes are ubiquitously distributed throughout the plant kingdom, and copal is one of its sources. They exist in a free form as aglycones or in combined forms, and have long been known to have a number of biological effects (Ikeda, 2008). The compounds: α -amyrin, βamyrin and lupeol, found in copal resins, have many bio-active properties.

Lupeol, a phytosterol and triterpene, is widely found in edible fruits, and vegetables. Extensive research over the last three decades suggest that lupeol has potential as an anti-inflammatory, anti-microbial, anti-protozoal, antiproliferative, anti-invasive, anti-angiogenic and cholesterol lowering agent [START_REF] Rahman | Beneficial effects of lupeol triterpene: A review of preclinical studies[END_REF]. Other properties of lupeol include a positive effect over conditions such as arthritis, diabetes, cardiovascular ailments, renal disorder, hepatic toxicity, microbial infections and cancer [START_REF]Pharmacological studies of various extracts and the major constituent, lupeol, obtained from hexane extract of Teclea nobilis in rodents[END_REF]Fernández et al, 2003;[START_REF] Rahman | Beneficial effects of lupeol triterpene: A review of preclinical studies[END_REF]. The available literature suggests that lupeol is a non-toxic agent and does not cause any systemic toxicity in animals at doses ranging from 30 to 2000 mg/kg [START_REF]Pharmacological studies of various extracts and the major constituent, lupeol, obtained from hexane extract of Teclea nobilis in rodents[END_REF][START_REF] Rahman | Beneficial effects of lupeol triterpene: A review of preclinical studies[END_REF][START_REF] Dzubak | Review Pharmacological activities of natural triterpenoids and their therapeutic implications[END_REF].

Ursolic and oleanolic acids have been investigated for their hepatoprotective effects. The mechanism of effect is complex. It includes suppression of enzymes that play a role in liver damage such as cytochrome P450, cytochrome b5, CYP1A and CYP2A, and an increase in antioxidant substances such as glutathione, metalothionein, zinc, glutathione-S-transferase and glucuronosyltransferase, with simultaneous protective effects on liver mitochondria.

Concerning anticancerous effects the first study, centered on oleanolic acid, was published by Hunan as early as 1995 when Pesha et al reported cytotoxicity of betulin acid onto human melanoma cell lines. To our days the spectrum of cytotoxity has enlarged to large lung cell cancer, epidermic carcinoma, melanoma and small cell lung cancer [START_REF] Dzubak | Review Pharmacological activities of natural triterpenoids and their therapeutic implications[END_REF]. Its efficiency against cell line proliferation has been probed [START_REF] Jing | Boswellic acid acetate induces differentiation and apoptosis in leukemia cell lines[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].

Betulinic acid has inhibitory action against HIV replication in H9 lymphocytes with an EC 50 = 1.4 µM and a therapeutic index (TI) = 9.3 (Yu, 2007). Synthetic derivatives of these molecules are under study for the treatment of these diseases.

CHAPTER I GENERAL PRESENTATION OF COPALS

PAOLA LUCERO

30

These compounds had the advantage of being lipophylic, which allows them to cross the bloodbrain barrier. In the ursolic family, ursolic acid for instance has the potential of an antiproliferative agent, because of its inhibitory activity against DNA polymerases α and β. It also has radioprotective effects in hematopoetic tissue [START_REF] Dzubak | Review Pharmacological activities of natural triterpenoids and their therapeutic implications[END_REF].

Concerning the oleanane families and their derivatives, it is widely accepted that boswellic acid have an effective anti inflammatory and ant arthritic effects, that have been profited in Indian Ayurvedic medicine.

On the other hand, αand β-amyrin acetates, ursolic and 23-hydroxyursolic acids have analgesic properties (PD 50 = 18.6-50 mg kg -1 ), which are comparable and in one case, even outclass the activity of acetylsalicylic acid [START_REF] Dzubak | Review Pharmacological activities of natural triterpenoids and their therapeutic implications[END_REF].

All the chemical, botanical, taxonomical, physical data that had been described until now in this work, were studied and used as a departure point for the collection of the samples, that will be present in the next chapter and that constitute the core of our research.

Chapter II

32

Chapter II Presentation of the copal samples and the analytical strategy

Extraction of copal

Studying the process of collection of copal in Mexico, implicates the study of a millenary technique [START_REF] Cruz-León | Antecedentes y actualidad del aprovechamiento del copal en la sierra de Huautla[END_REF]. The survival of this practice is related with cultural motivations, as copal is intimately linked with ritual pre-Columbian practices that did not disappear upon the contact with European culture.

To this respect one can cite the Mendoza codex also known as "Matrícula de tributos". This document was elaborated immediately after Spanish conquest, and details the Aztec empire´s administration, with the name of the over 30 provinces under Aztec domination and the taxes that should be paid. It contains information about the type of product, the amount and the frequency of tribute merchandise. Concerning copal this codex mentions "400 small baskets of white copal and 8000 small balls of refined copal" to be paid annually.

The actors of the collection of copal are known as "copaleros", in our days there are few people dedicated to this activity. They work in the collection of copal in the season comprised between July and September from each year.

The knowledge on the techniques of extraction of copal, on the conservation of the trees and the selection of them constitute a heritage from a generation to another. Usually the "copalero" is assisted by a son or a grandchild from young age.

A study performed in the region of Morelos [START_REF] Cruz-León | Antecedentes y actualidad del aprovechamiento del copal en la sierra de Huautla[END_REF] showed that each "copalero" may collect resin from up to 200 hundred trees in each season. During collection, the journey last eleven or twelve hours.

For the extraction of copal, some cuts are practiced perpendicularly to the tree trunk, in the phloem. Then the resin is collected in leaves of maguey (Agave angustifolia). The size of maguey leaves goes from 35 to 45 cm long and 3 to 8 cm width. This size is related to the bars of copal that can be bought at commercial markets.

In American context, «copals» are collected from Bursera or Protium species from the Burseraceae family (Becerra and Venable, 1995). The quality and color of the copal depends on the tree species from which is extracted (figure 18), when collected it is a translucent liquid that shortly after turns into yellow, white or amber solid (García-Hernández, 2000). The technique of harvesting copal also has an impact into the final commercial product.

While white copal is the main product, exuded directly from the incisions practiced in the bark of the tree, "copal piedra" is the result of a defense reaction against the attack of an insect: Chyptodes dejeani (Montufar, 2007).

The "lágrima copal" (copal in tears) is the product remaining in the recipient of collection and the incision at the bark of the three (Cruz-Leon, 2006).

The section of the bark that was cut upon the extraction of the resin and, that is fully impregnated with it, is also collected in Mexico. In the traditional markets it is sell as "Mirra" which has no relation to the actual myrrh (as it has been said previously on this work, it is produced by another tree species corresponding to Commiphora myrrha). In figure 19 are presented some pictures of the different kinds of copal, found in traditional markets from Mexico. (1982), in Guizar-Nolazco and Sanchez-Velez (1991) and in [START_REF] Johnson | The genus Bursera in Sonora, Mexico and Arizona[END_REF]; the last two woks also has ilustrations.

B. laxiflora S. Wats (B. concinna Sandw.) description in [START_REF] Johnson | The genus Bursera in Sonora, Mexico and Arizona[END_REF].

B. penicillata (DC.) Engl.(B inopinata Bullock). Description in Bullock (1937, under the name of B. inopinata) and in Rzedowski and Guevara Fefer (1992). Ilustration in [START_REF] Mcvaugh | Synopsis of the genus Bursera L. in western Mexico, with notes on the material of Bursera collected by sesse & Mocifio[END_REF].

B. simaruba (L.) Sarg. (B gummifera L.) Description in Toledo Manzur (1982), Rzedowski andGuevara-Fefer (1992), Rzedowski and Calderón de Rzedowski (1996b) and [START_REF] Rzedowski | Las especies de Bursera (Burseraceae) en la cuenca superior del rio Papaloapan (Mexico)[END_REF]; ilustration on the last work.

B. stenophylla

Sprague & Riley. Description in [START_REF] Riley | Contributions to the flora of Sinaloa[END_REF] and [START_REF] Johnson | The genus Bursera in Sonora, Mexico and Arizona[END_REF]; the last work includes illustrations.

For the collection of the certified origin samples this research counted 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 body of Biomaterials from the UAS (UAS CA-208).

The collection proceeded from summer to fall, as in these times of the year trees have both leaves and fruits, which are necessary for botanical identification. After practicing a small cut into barks tree, resin was collected in glass tubes.

CHAPTER II

PRESENTATION OF THE ANALYTICAL STRATEGY AND OF THE COPAL SAMPLES

PAOLA LUCERO

35

The criteria for the discrimination of the tree species was based on the bibliographical works mentioned at the beginning of the present section. A research work containing detailed information on this subject and a in-depth approximation to botanical aspects is under preparation of Dr. Rito Vega. As an example reference leaves showing the differences between leaves of B.bipinnata and B.stenophylla can be consulted in annex 1, table 37.

Choice of the studied species

Regarding the inclusion of each species in the study, our choice was oriented by several factors that will be discussed below.

We include B. bipinnata as its pollen has been founded by paleobotanist, in archaeological context in the site of Templo Mayor (Montufar, 2007). B. bipinnata is a species with a wide geographical presence and sometimes it produces hybrids with other members of the Bullockia section. As the botanical distinction between B. stenophylla and B. bipinnata is unclear, we decided to include this second specie as well (Rzedowski, 2005).

As it has been said on section 5.1.1 of chapter 1, Bonampak murals from the Maya Culture had been studied and researchers conclude that the gum of B. simaruba might have been used

as agglutinant [START_REF] Magaloni | Los pintores de Bonampak[END_REF]. So we include this specie to contribute to the knowledge of the chemical composition of this oleo-gum-resin.

As B. grandifolia is a well defined Bursera tree and it is related to B. simaruba, we included it in the study to compare its chemical composition to the first one.

It is important to note that the genus Bursera has been divided into two subgenera or sections: Bullockia and Bursera [START_REF] Mcvaugh | Synopsis of the genus Bursera L. in western Mexico, with notes on the material of Bursera collected by sesse & Mocifio[END_REF]Rzedowski, 1968;Becerra, 1997). B. simaruba and B. grandifolia belong to the section Bursera (subgenus Bursera), while the rest of the studied species belong to the section Bullockia [START_REF] Rzendowski | Inventario del conocimiento taxonómico de la diversidad y del endemismo de las especies mexicanas de Bursera (Burseraceae)[END_REF]. See figure 3 chapter 1 of this work.

Colloquially B. simaruba, B. grandifolia and the other species from the subgenus Bursera are known as "cuajiotes" a Nahuatl word which means leprous tree and that refers to a botanic characteristic of this subgenus, as tree bark exfoliates [START_REF] Becerra | Nuclear ribosomal DNA phylogeny and its implications for evolutionary trends in mexican Bursera (Burceraceae)[END_REF].

To have an idea on the variability of the chemical composition of Bursera resins from Mexico, other three species were chosen: B. excelsa, B. laxiflora and B. penicillata wich are not related to the other 4 species. Only one sample of resin from B. copallifera was collected, this sample was studied by all the analytical techniques described in the strategy for this For orientation along this work, we included in annex 1 (tables 27, 28 and 29) data of certified resins concerning: the tree species, its GPS localization with a map and the date of collection.

For selected cases of commercial resins, pictures were also included.

3 Archaeological samples from Templo Mayor Site

In table five we present information corresponding to each studied archaeological sample from this site. It concerns the number of artifact from which sample was taken as well as the number of offering where artifact came from.

The nature of the samples was variable: sample 84 was taken from the adhesive material found in the base of a ceremonial knife, sample M1 was part of the molding material of a In further sections of this chapter the offerings, the physical location and the physicochemical conditions that surrounded these samples for over 500 years are described. After excavation the conservation treatments that they were subjected to, are described here as well. This information was taken from activities report of conservation and restoration of the collection of archaeological materials, 7 th season (Alonso A. et al, 2008).

Description of the offerings from Templo Mayor

The offerings deposed in the Templo Mayor of Tenochtitlan are constituted by sets of objects carefully ordered. Some of them were placed in stone boxes underneath the buildings, whereas others were buried directly in the constructive filling material between constructive The garments found in the offerings may be identified to the Aztec priesthood. Some offerings include vestiges of flora and animals. Between the identified fauna there are crocodiles, jaguars, pumas, lynxes, serpents, as well as a great diversity of birds, fish, shells and marine snails (Lopez-Lujan, 2009) and a wolf in the case of offering 120. A picture of one of the levels of these offerings is presented on figure 22. The variety and abundance of objects may be explained by the importance of the non verbal communication among the Aztecs: olfaction and vision, played a key role in their rituals of communication with the Gods, for instance only Tezcatlipoca could smell the scent of the flowers in an offering (Hayden, 1980).

Therefore these characteristics of the offerings opened a new line of investigation around Mexica's Cosmovision. One of most interesting, approaches lies in the relation between objects distribution and the meaning of the offerings. [START_REF] Jimenez-Badillo | Una aplicación de la teoria de gráficas en arqueología (primera parte)[END_REF]. Previous investigations (López Lujan, 1993 and1997;Lopez Austin, 2004;[START_REF] Contel | el cerro, la olla y el chalchihuitl. Una interpretación de la lamina 25 del codice Borbonico[END_REF][START_REF] Jimenez-Badillo | Una aplicación de la teoria de gráficas en arqueología (primera parte)[END_REF] concluded that Mexica priest, that were in charge of the composition and deposition of the offerings, used a very sophisticated symbolic code to communicate with Mexica gods.

This code determined not only the location of each element of the offering, but also the categories of objects that had to be combined in order to express a specific concept. For instance, in some deposits, the image of the God of the fire was located next to other objects symbolically related to the "hot" portion of cosmos. At the same time, these sets were in symmetrical opposition to objects with "cold" connotation, the former ones related to God of rain and the fertility. An obvious interpretation of this code is that it expresses the concept of Another appellant and recurring arrangement of several offerings is the positioning of an object in the center and four more in the corners. This seems to be a reference to the geometric model of the Mexica universe, whose scheme included the center of the world and the four cardinal directions. (Jimenez-Badillo, 2009) A clear example of both schemes is founded in offering U (figure 23). These cosmic symbols turn the offering into an authentic scaled image of the Universe, where the lower levels represent the different infra world spheres and the higher ones the celestial spheres (Lopez-Lujan, 2009). Consequently, when approaching the study of these offerings it is precise to consider in one side the type and variety of objects that contain, and on the other side their distribution in the space.

Considering the artifacts in the offerings, physical and chemical factors play a key role into degradation processes: the compression of the materials owed to gravity, the erosion generated by the contact with abrasive particles, the fluctuations in water level (as the site is placed on a water source) that inundated the offerings and its partial evaporation, the pH of this water and the action of biological agents, the temperature and the concentration of oxygen, had a major impact into the differences that arise in the preservation state of archaeological objects from buried deposits.

CHAPTER II

PRESENTATION OF THE ANALYTICAL STRATEGY AND OF THE COPAL SAMPLES

PAOLA LUCERO

41

The archeology in Mexico City faces the same challenges than in Rome, Jerusalem or Istanbul: it is constrained to the circumstance that ancient and highly valuable remains of a sophisticated civilization lye under the ground of a modern city (Lopez-Lujan, 2010).

The particularity of Mexico remains in the fact that, at this particular moment of the history, is the most populated city in the world and at the same time, it hoards the richest set of historical monuments of American continent (Lopez-Lujan, 2010).

Macroambiental conditions

Mexico City is located at a height of 2240 meters above sea level; the weather is temperate with two seasons along the year: a dry one and a rainy one, the last one, going from May to October. The difference of humidity between the two seasons is of about 40%.

Along Mexico runs the volcanic transversal axe thus, Mexico City is a telluric zone.

Earthquakes related to volcanic activity are common, and may produce harm onto buried elements as in urban pipelines of the network of distribution and water collection.

Templo Mayor is placed, on a desiccated lacustrine soil, where the freatic nap is located close to the soil surface.

The ground is composed of clay and lime that interact with water, allowing the mobility of materials and a differential compactation (Alonso et al, 2008). This type of soil is permeable and allows the dragging of salts, pollution and clay onto the interior of offerings.

Concerning the buildings surrounding the site they have considerably different foundation systems: some colonial buildings were erected on the remains of Aztec buildings, while some others were constructed with Spanish technology. All the enumerated factors contribute to the sinking and/ or inclination of some areas.

As the site is located within an urban emplacement it is subjected to the presence of air pollution, and in some cases acid rain. Global warming had impacted the site as well, as ground temperature has certainly been increasing through the last years.

Microambiental conditions of Templo Mayor offerings

Offerings 120, 125, 126, 128 and 140 were excavated in what was once a colonial building known as "Casa de las Ajaracas"or in the esplanade just before this building. The principal stairs of the sixth constructive stage of the Templo Mayor were constructed in the physic space of this lot, as well as the esplanade in front of the temple, and the vestiges of what is supposed to be the Cuauhxicalco: the temple where Aztec emperors were cremated (figure 24).

These offerings are associated to the Tlaltecuhtli monolith, a superb piece of lapidary Aztec art, of 3.57 for 4 m long andesite, carved in relief. It was partially stuccoed, painted in red, ochre white, black and blue (figure 25). Generally speaking objects from the mentioned offerings that are manufactured in copal present heterogeneous states of preservation. A tendency towards a yellow coloration is verified with ageing. More than the half of the unburied copal is fragmented, its exterior is dusty and the surface presents layer or flake separation.

2.4

Description of archaeological Aztec offerings

Offering 120

It was excavated from June 2007 to November 2008.

It corresponds to the VI th constructive stage of Templo Mayor (built between the years 1486-1502 A.D.). The offering was placed inside a wrought stone box, the walls were constructed with flat smoothed blocks of andesite and a seal of the same material. When it was excavated the archaeological objects that constituted it, had a high content of water but it was not flooded.

Nevertheless during some months during the excavation the offering was flooded due to broken pipelines of the public service of water. The nature of water was potable but also from sewing.

Its pH was 7.82.

The presence of sewage water had a high impact onto copals within the offering: their surface was blackened.

Among the materials founded into the offering are: bars of copal with fibers of maguey onto their surface, maguey thorns rests of basketworks and hard fibers, objects of different materials like shell, bone, alloys of copper, flint, obsidian stone and green marble.

CHAPTER II

PRESENTATION OF THE ANALYTICAL STRATEGY AND OF THE COPAL SAMPLES

PAOLA LUCERO

41

Comparatively speaking to the general state of preservation of the objects in this offering, it is inferior to the one of the objects of offerings 125 and 126.

The copal from the offering 120, was stick to maguey bars, are light yellow and brown colored, owing to the presence of organic material that polluted the context.

Conservation and Restoration Treatments

The copal from this offering was cleaned, dry under controlled conditions, consolidated, when it was possible fragmentized pieces were joint, and then it were fumigated, finally they were conditioned for its storing.

Offering 125

It was excavated from April 2008.

The offering was found in a tunnel constructed with polished tezontle walls, sealed under two other offerings, between different seals of stone and stucco. From the entrance of the tunnel to the offering there are 2 m and a half. This situation created a good preservation environment with stable conditions of temperature, light, oxygen and elevated ambiance moisture of around 92%, which allowed the conservation of a lot of organic materials such as wooden objects, seeds, animal hair and bones, basketworks, maguey thorns, copal and "guajes" (which are the dried fruits of Lagenaria siceraria, and were used as liquid containers by prehispanic cultures).

Among the remarkable inorganic materials from this offering are the turquoise tesels associated to the ornament of a pedernal knife and golden bells and ornaments from the wardrobe of the representations of Gods.

The objects were covering by sedimentary clay, and decomposed organic material.

During excavations some potable water infiltrations that affected the offering were registered, causing its flooding. This water had high content of chlorine and zinc, therefore water content was drained from the offering.

Conservation and Restoration Treatments

The copal from this offering was cleaned with soft brush and water to wash out the sedimentary layer that covered it. As the offering 125 and 120 it was flooded and the pH of the water was between 6.8 and 7. A particularity that is worth to mention, is that water presented and oily phase that may be owed to the dissolution of the essential oils from the copal bars; copal was very abundant in this offering, along with marine and botanical remains.

Objects were covered with a thin layer of sedimentary clay of 3 to 4 mm thickness.

This offering was colonized by black and white millipedes and fungi so it was subjected to a biocide treatment.

Conservation and restoration treatments

The copal from this offering followed a very similar process than the one from offering 120: it was cleaned, dry under controlled conditions, consolidated; when it was possible, fragmented pieces were joint, fumigated and finally conditioned for its storing.

Offering 128

This offering was found to the east and under the level of offering 125. The offerings are comprised into the constructive filling between two stages of the temple. It was located 12 m under the ground level. It was flooded, as the filling material is made of clay and lime and the water nap is located close to the surface. In this case the weight of the superior layers had produced harm owing to compaction.

Owing to its deep localization fluctuations of temperature and humidity were minimal, and concentration of oxygen was also limited.

Archaeological sample from the "Sacred Cenote" of Chichén Itzá

The [START_REF] Pedroza | Cenotes y cuevas inundadas de la península de Yucatán: de los primeros pobladores a la Guerra de las Castas[END_REF].

Its current morphology is the result of a series of geological and geomorphical events: karstic rock is highly permeable, allowing therefore the infiltration of meteoric water. This water cumulates in the underground, floating over a mass of marine water. The process of filtration together with changes in temperature the collapse, construction and dissolution of carbonates, contributed to form the biggest system of submerged caverns of the world, shelters, dolines, rivers and "cenotes", [START_REF] Beddows | Los cenotes de la península de Yucatán[END_REF] cf figure 26.

The word "cenote" is a regional, mayan term derived from the word ts'ono'ot or d'zonot that means fresh water lake or cavern with deposed water [START_REF] Rojas-Sandoval | Cementerios acuáticos mayas[END_REF]. In fact this term [START_REF]Los cenotes en la actualidad: entre la veneración y la explotación[END_REF].

It is important to note that the only source of fresh water in the whole peninsula is that of "cenotes", the "Hondo" river being the only source of superficial water in the entire peninsula of Yucatán. Not all the cenotes in Yucatán present archaeological remains on its depth. Which demonstrates that among ancient Mayas two types of cenotes existed: for water supply and for ritual use. For the Maya people, caverns and water bodies were the symbolic entrance to infra world and cenotes were the home of the Chaacs Gods of the rain. Therefore these sites were object of veneration [START_REF]Los cenotes en la actualidad: entre la veneración y la explotación[END_REF]. They were also considered as well as places of born and dead [START_REF] Rojas-Sandoval | Cementerios acuáticos mayas[END_REF]. Soon after their arrival Cenotes caught the attention of the Spanish, who, amazed at their singularity, devoted space to describing them in their chronicles and accounts.

Cenotes can be classified according to their geological, hydrological, biological and chemical characteristics, so that we speak of 'young' or 'lotic' cenotes, and 'old' or 'lentic' cenotes. The first ones communicate freely with the aquifer through the galleries of the cavern, so that the water flow is horizontal and the water is present for only a very short time. Consequently, there is little organic matter as sediment and or in suspension.

In the lentic ones, the cenotes experience a blockage caused by the collapse of walls over centuries, and by natural sedimentation processes. These factors restrict exchanges with underground waters and makes refilling much slower. In this case there is sedimentation and

45

dissolution of particles of organic matter and detritus, which has a direct impact on the water's biological, physical and chemical processes.

While water clarity is a feature of young cenotes, the water in old cenotes tends to be turbid because of the presence of suspended particles. In fact, the production of organic matter in a cenote depends on the degree of opening or exposure of the aquifer to the outside and on the incidence of light. Such characteristics must of course be taken into account in underwater exploration work.

Microambiental conditions

One of the more appealing and outstanding features in Chichén Itzá is the Sacred Cenote or Cenote Chenkú [START_REF] Cobos | El cenote sagrado de Chichén Itzá[END_REF]. It is located at the end of Sacbé 1 (avenue 1), 300 m to the north of the pyramid known as the Castle. It is a circular cenote of 60 m diameter and the maximal depth of its water is 13.5 m, cf figure 27. The distance from the ground surface to its water is 22 m depth, its walls are vertical and it belongs to the older or "lentic" type. Chichén Itzá Sacred cenote being a closed system, has no water streams, therefore an accumulation of organic and particled material is observed [START_REF] Beddows | Los cenotes de la península de Yucatán[END_REF]. The amount of organic material deposed onto this cenote depends directly onto its diameter.

Owing to this organic material accumulation, there is low penetration of UV-Visible light, exposition to direct sun light takes place only at noon each day [START_REF] Rojas-Sandoval | Cementerios acuáticos mayas[END_REF].

The amount of oxygen solved in the water and the pH decreases with the depth of the water of the cenote [START_REF] Beddows | Los cenotes de la península de Yucatán[END_REF]. These factors along with the turbidity of its water, its low, and its fine sediments allow an extraordinary preservation of archaeological evidence.

CHAPTER II

PRESENTATION OF THE ANALYTICAL STRATEGY AND OF THE COPAL SAMPLES

PAOLA LUCERO

46

There are two periods of height of the Sacred cenote as a worship place in the Classic (800-1100

A.D.) and the Postclassic (1100-1550 A.D.).

During the Classic Chichén Itzá dominated a great portion of the north of the Yucatán peninsula.

The source of its power was the long distance commerce of different merchandises.

It is important to note that among all the cenotes in Yucatán, Sacred cenote is extraordinary because there is no other cenote, with such a variety and number of archaeological objects and offerings. Among the materials recover from its bottom can be found gold, jade, copper, textiles and of course copal [START_REF] Rojas-Sandoval | Cementerios acuáticos mayas[END_REF].

The origin of these objects can be related to the current territories of Guatemala, Costa Rica, Panamá, the south-east of the United States, and of course the Yucatan peninsula.

Underwater Chichén Itzá cenote explorations

Along the XIX th century many explorers and adventurers with no actual formation to the archaeology were attracted to the cenote. This attraction was due in good part to the XVI th century chronicle "Relation of the things of Yucatán" from Diego the Landa.

In 1882, the eccentric French explorer Désiré Charnay tried to dredge the cenote with a Toselli sounder. However, the irregular bed of the well, the presence of stones, trunks and roots prevented the explorer from extracting anything, so he gave up (Martos- [START_REF]Los cenotes en la actualidad: entre la veneración y la explotación[END_REF].

In 1894, E. Thompson the first consul of the United States in Yucatan bought the Hacienda of Chichén Itzá. This hacienda included the remains of the ancient city and the cenote. From this year and up to 1907 the cenote was systematically dredged.

Between 1909 and 1911, Thompson changed its dredging technique for a diving one. In all the recover more than 30 000 objects (figure 28) among them where: ceramic vessels, carved jade beads with inscriptions, obsidian and quartz, figurines, shells, gold plated copper cups, gold bells, discs and pendants and human bones. Many of these vessels were filled with copal (figure 28i and 28j), and there were found also figurines moulded in copal, shaped as faces, corn, etc (figure 28h and 28k to m), [START_REF] Cobos | El cenote sagrado de Chichén Itzá[END_REF].

The bells along with wooden lances with flint ends, witness of a first identifiable phase of offerings associated with a warrior cult.

Commercial samples

The interest of studying commercial copals is that these materials are traditionally used by artist in paintings but also for restorers, solved with ethanol as adhesive (personal communication from Maria Barajas). Among the painters that included copal in their paintings are Diego Rivera and Murillo (De la Cruz Cañízares, 2005).

Traditional markets in Mexico can be classified in two categories: the seasonal ones, that are associated with religious festivities and, that are settled in a location only for few days and the permanent markets like the Sinaloa and Azcapotzalco markets in Mexico City or the Tepoztlán market in Morelos.

"Copaleros" usually sell their products in seasonal markets were merchants from permanent markets buy copal for the year.

For this study commercial copal was collected by the author of this work, from seasonal markets in different locations like: Izúcar de Matamoros and Cholula in Puebla, Huitzuco in Guerrero and, from Azcapotzalco and Sonora permanent markets in Mexico City.

Dr. Lauro Bucio, and Dra Irma Belio also collected copal from Oaxaca, Colima and Estado de Mexico.

In this way the geographical zone of collection of commercial copals cover a good part of the geographical zone that was once Aztec territory (figure 29).

CHAPTER II

PRESENTATION OF THE ANALYTICAL STRATEGY AND OF THE COPAL SAMPLES

PAOLA LUCERO

49

It is important to note that in traditional markets, resins from other botanical origin than Bursera may be found: for instance "zumaque" (Rhus sp.) and "estoraque" (Liquidambar styraciflua), a black copal looking resin (Myroxylon balsamum) among others [START_REF] Montúfar | Los Copales Mexicanos y la resina sagrada del Templo Mayor de Tenochtitlan[END_REF]. Their differentiation only by organoleptic analysis (physical charcteristics) being impossible (Régert, 2008). The physical characteristics of the samples are present on tables: 4, 5 and 6. Samples were collected on glass tubes after a small incision had been made in the tree.

Presentation of the analytical strategy

Based on the principle that the knowledge of the art and archaeological objects should be based on the examination of the artwork itself (Domenech-Carbó, 2008) an analytical strategy was created. This approach was necessary, in this case as the knowledge of the manufacturing of aircrafts in Ancient Mexico is quite ambiguous and most of the time is fragmentary and scarce, due to the lack of documentary sources.

Even more studying the chemical composition of organic biomaterials associated to archaeological artifacts can give an important contribution to archaeological, conservation and ethnographic studies. Additionally the molecular analysis of the residues of these copal materials can answer questions relating to their botanical origin, their collection and preparation procedures, and their geographical origin. Chemical diagnosis helps to establish the extent and nature of degradation of the materials, important factors in conservation and restoration.

As it has been said in section 5 of chapter 1, the study of natural products-as resins -may be difficult owing to the complexity of these materials. The stability of an organic compound depends on its chemical structure, which determines the alteration mechanisms observed in organic materials. In consequence no general analytical technique exists to identify these substances and their degradation products, because they contain the same common elements (Colombini, 2000).

Nevertheless analyses on archaeological samples are difficult given the restrictions imposed on the number and size of the samples. Therefore the creation of an analytical strategy was necessary. A schematic view of the situation is presented on figure 30.

In this strategy certified origin samples and commercial samples were considered as reference materials to study the behavior of ancient copal.

A series of test was performed in a parallel way in both historical and fresh resins: the first step of the series was to perform a detailed visual examination, in both macro and microscopically levels. As archaeological samples exhibit a heterogeneous nature a stratigraphical analysis was performed. This analysis made evident an accelerated deterioration process on the surface of some archaeological samples.

The second step of this strategy consisted in a FTIR analysis of all samples. Spectral fingerprints for each botanical origin in certified samples were established.

The third stage in the strategy consisted in the HPLC/UV-Vis analysis of the samples, molecular differences in composition of resins from certified origin were remarked.

In order to achieve a better interpretation of both FTIR and HPLC-UV-Vis data two statistical methods were applied: PCA (Principal Component Analysis) and LDA (Linear Discriminant Analysis). The first one is a descriptive mathematical method which can help to recognize patterns, it produces a graphical were data is positioned according to their description. LDA is a classification method that uses linear combinations to minimize intra-species variations at the same time that it maximizes interspecies variations. Results can be consulted in Chapter III and IV of this work.

The next step in the analysis consisted in the GC-MS analysis focused on the triterpenic fraction of the resins. This highly sensitive technique allowed identifying molecular markers for each studied resin species, at the same time that it opened new perspectives in the understanding of aging phenomena.

The last step of the strategy consisted in the consideration of all results to characterize effectively the origin of archaeological samples, as well as the changes experiences by copal under natural and accelerated ageing.

This strategy is based in the interpretation of analytical results based on artistic, archaeological, iconographic and iconological considerations following the model proposed by Tchapla, 1999.

Study by optical microscopy

In order to obtain the maximum information from one sample, a systematic preliminary analysis by optical microscopy on cross sections was performed. For some selected samples a study by electronic microscopy was performed, their nature is quite heterogeneous, some selected pictures of these results are shown on annex 1 (table 31 to 35) of this work.

Concerning archaeological samples, a first approach by optical microscopy allowed us to establish the heterogeneous nature of some of them (figure 31).

The microscopic examination of these samples revealed the presence of multilayer in archaeological samples, a difference in density between the outside of the samples (subject to a more intense ageing) and the inside was noticed: the inner part of the samples therefore showed to be much less deteriorated. Variations of color towards yellowing and/or blackening, lost of transparency and greater porosity, were also noticed on the surface. In sample no 26, two phases were observed: a white opaque one and a darker, amber coloured bright one.

Some other samples like M1 and 51, 52 were quite homogeneous, with no amber phase but instead some black dots on a whitish matter (figure 32).

Also remarkable is the closeness of aspect of samples 51 and 52. This fact can be explained considering that both samples come from the same archaeological offering, and therefore they were subjected to the same ageing conditions.

CHAPTER II

PRESENTATION OF THE ANALYTICAL STRATEGY AND OF THE COPAL SAMPLES

PAOLA LUCERO

A)

Size: 3mm x 2mm 80x

B)

Size: 3mm x 2mm 80x

C)

Global size 2 cm 80x

Fig 32 Homogeneous archaeological samples A) 51, B) 52, with inclusion of hair C) M1

Sample TMT exhibit a very deteriorated and blackened surface, while the interior of the sample was white and brilliant as it was the surface of fresh resins (figure 33). In consequence, partial analysis -by FTIR and GC/MS-of the different phases were performed when possible. Then the result was compared to that of global analysis, to assess degradation products, and the quality of the information obtained from deteriorated surfaces.

Conclusion

Even if the contribution of optical microscopy is modest to the results of this research, its utility is undeniable. It allowed us to distinguish a difference between the state of conservation of the surface of archaeological samples and its inner part.

In aged samples important physical changes could be observed. In fact along ageing resins oxidize by a mechanism of radical formation in a chain of events that begins with the attack of oxygen to double bonds, present in triterpenoids [START_REF] Dietemann | Towards more stable natural resin varnishes for paintings[END_REF]. These phenomena may lead to the formation of new insaturations that may act as chromophores, which yellow or darken resins appearance [START_REF] De La Rié | Photochemical and thermal degradation of films of dammar resin[END_REF]. At the same time these insaturations due to oxidation, enhanced resin sensibility to radiation and humidity, contributing to cracking and oat formation that were observed at the surface of some samples, all these phenomena were observed in archaeological resins, to different extents.

Degradation state of resins is connected to both original composition of the material and to ageing conditions, as ageing seems to be more intense in the surface of the resin a partial study of these more deteriorated parts will be performed, as part of this study. A number of papers have identified natural materials by means of chemometric approaches [START_REF] Tapp | FTIR spectroscopy and Multivariate Analysis can distinguish the geographical origin of extra virgin olive oils[END_REF][START_REF] Rezzi | Composition of Pinus nigra ssp[END_REF][START_REF] Armanino | Modelling aroma of 3 italian red wines by head space-mass spectrometry and potential functions[END_REF]Casale, 2010a, Casale 2010b). In the artistic field, the most common is PCA (Principal Component Analysis), which has been applied to the discrimination of proteinaceous media (Andreoti, 2006;[START_REF] Colombini | Two procedures for suppressing interference from inorganic pigments in the analysis by gas chromatography-mass spectrometry of proteinaceous binders in paintings[END_REF][START_REF] Gautier | GC-MS identification of proteins in wall painting samples: A fast clean-up procedure to remove copper-based pigment interferences[END_REF]Bonaduce, 2006), seccative oils (Andreoti, 2006;Comlombini 1999) polysaccharides [START_REF] Colombini | Ion chromatography characterization of polysaccharides in ancient wall paintings[END_REF] and resins (Vieillescazes, 1995).

Up to now, characterisation of natural resins has been based on the use of specific markers, which can vary depending on materials ageing [START_REF] Mills | The gas chromatographic examination of paint media. Part I: Fatty acid composition and identification of dried oil films Studies in Conservation 11 (1966) 92-Mills J.and White R. Natural resins of art and archaeology: their sources, chemistry, and identification[END_REF]White, 1977 and1987) being this a disadvantage in case of degradation of molecules. As it has been said composition of plant resins is quite complex and is not entirely known yet for the majority of plant species (Colombini, 2000).

59

The objective of this technique is to capture the intrinsic variability in the data at the same time that reduces the dimensionality of a data set, either to ease interpretation or as a way to avoid over fitting and to prepare for subsequent analysis [START_REF] Terradez | Principal component analysis[END_REF].

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 [START_REF] Cosio | Evaluation of different storage conditions of extra virgin olive oils with an innovative recognition tool built by means of electronic nose and electronic tongue[END_REF].

The aim of principal components analysis is to explain the maximum amount of variance with the fewest number of principal components (the smallest lost of information). It allows to consider the preferred directions of the data and to ignore those variables that do not have a big influence in the separation of the groups (Barreto, 2008). When data is projected in two dimensions (or three), 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 a uniform behaviour to each other and different from other existing groups [START_REF] García | Modelización por técnicas no paramétricas del proceso de mezclas de gomas para extrusión[END_REF].

PCA can be used to reduce the number of variables when there are too many predictors relative to the number of observations.

PCA is one of the most used classification techniques: and to sum up is a probabilistic parametric classification technique that maximizes the variance between categories and minimizes the variance within categories, by means of a data projection from a high dimensional space [START_REF] Ferré | Selection of components in principal component analysis: a comparison of methods[END_REF].

Linear discriminant analysis (LDA)

LDA is a powerful chemometric tool that allows finding a predictive classification model, meaning that allows the construction of model able to foretell the property of a sample to a category previously defined, or to investigate how 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 if two or more populations are different from each other;

and classification in strict sense, where given two or more populations and an object that belongs to some of them, it finds out to which of them it corresponds. The discriminating analysis requires to obtain combinations linear (or nonlinear) of independent variables that will discriminate between defined groups a priori, so that the errors of the bad classification must be minimum [START_REF] Barreto | Minería de datos aplicada a la mejora de procesos de extrusión de elastómeros[END_REF].

The LDA model is constructed from a set of samples of well-known category, denominated training data set, and then a regression equation is used as discriminating function (Casale, 2010 a).

Once the model is obtained, this one is used for the prediction of the category of property of the samples of unknown category. LDA can be linear or quadratic. The linear one 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 is removed once 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 [START_REF] Lachlan | Discriminant analysis and statistical pattern recognition[END_REF].

The quality of the LDA classification model was considered on the basis of the validation results.

The statistic analysis (PCA and LDA) in this work was performed using Minitab 16 (Minitab Inc. PA, California).

Chemotaxonomy. Correlation of resin composition and botanical origin

Botanical systematic or plant taxonomy is the science of delimiting, describing or naming of, any group of plants considered to represent a distinct unit (Hegnauer, 1985). In many instances phylogeny of taxa is reconstructed with the aid of character comparison of living plants. The task of taxonomists is rendered very difficult, however, by relatively rapid divergences of characters and taxa in the course of adaptive radiation and, by several types of convergence known to occur during taxon evolution. Inevitably all existing systems of classification are imperfect in part. In such cases chemical characters may be very helpful.

In its broadest sense, chemotaxonomy is the use of chemical diversity as a taxonomic tool [START_REF] Frisvad | The use of secondary metabolite profiling in chemotaxonomy of filamentous fungi[END_REF].

Plants are stunning chemical factories and, chemotaxonomy provide some broad assessments regarding the distribution of various structural classes of secondary metabolites such as alkaloids, quassinoids, flavonoids and triterpenes [START_REF] Cordell | Phytochemistry and traditional medicine. A revolution in process[END_REF]. These compounds may are called polytypic (Hegnauer, 1985).

The composition of plant resin is quite complex and, not entirely known yet for the majority of plant species, it may also depend on climate and on the characteristics of soil (Colombini, 2000).

Terpenoids and chemotaxonomy

Secondary metabolites are chemical compounds produced by a limited number of species in a genus, order or even a phylum. Usually they occur in low concentrations and are not essential to plant cell survival. It is assumed that their function or importance is mainly related to ecological aspects, as they are often used as a defense against predators, parasites and diseases, for interspecies competition and, to facilitate the reproductive processes (Shulz and [START_REF] Schulz | Identification and quantification of valuable plant substances by infrared and Raman spectroscopy[END_REF].

Terpens represent a large group of plant substance that occurr in flowers, stems, leaves, roots and resins. It has been found that considerable variability in terpenoid composition in under genetic control and therefore terpenoids can be often acts as biochemical markers for chemotaxonomic classification (Shulz and Baranska, 2007).

Therefore some of the main components of the oleoresin (mono-, sequi-and triterpenes) have been used when studying other species as chemotaxonomic indicators and biochemical markers for instance of provenance of Pinus [START_REF] Arrabal | Differentiation among five Spanish Pinus pinaster provenances based on its oleoresin terpenic composition[END_REF], Boswellia sp (Mathe, 2003) and dammar from Dipterocarpaceae [START_REF] Burger | Charactérisation moléculaire de résines végétales archéologiques et actuelles: Étude de résines de dipterocarpaceae[END_REF].

Study by Fourier-transformed infrared spectroscopy (FTIR)

In the context of cultural heritage, the use of non destructive diagnostic techniques such as infrared spectroscopy (FTIR) had been used since 1954. This tool is highly valuable, in fact is one of the most widely used techniques in this field [START_REF] Van Keulen | Qualitative effects of knowledge rules and user feedback in Probabilistic Data Integration[END_REF], because of its ability for providing structural information of both organic and inorganic materials.

The scope of classical IR spectroscopic analysis was extended by the incorporation on IR Fourier transform spectroscopy owing to its superiority in terms of signal-to-noise ratio and resolution, while only a extremely amount of sample is needed to perform the analysis (Doménech, 2008). Infrared spectroscopy is based upon the study of the absorption of a sample of electromagnetic radiation with a wavelength between 10 000 and 10 cm -1 . This range is divided itself into near infrared (from 10 000 to 4 000 cm -1 ), medium infrared (from 4 000 to 400 cm -1 ) and far infrared (under 400 cm -1 ) (Rouessac, 1998).

The richest part of the spectra and the most accessible from the experimental point of view is that of medium infrared. In this region, absorption under 800 cm -1 corresponds to a molecular fingerprint and, those over this wavelength are characteristic from the chemical functions present in the sample, which allow its analytical and structural analysis. In some cases the observation between the reference value and the experimental data allows to establish the surrounding environment of a given function in a molecule. For instance electronic delocalization and formation of hydrogen bonds shift down the absorption value [START_REF] Bellamy | The infra-red spectra of complex molecules[END_REF].

FTIR spectra of terpen and terpen like molecules

When dealing with natural resins, and being these ones constituted by many different compounds, it is impossible to perform a work structural identification by means of this technique [START_REF] Hovanessian | Différenciation de substances naturelles par diverses techniques analytiques: spectroscopie IRTF, CLHP / uv-visible / fluorimétrie et CPG /SM[END_REF].

Nevertheless according to a previous study (Shulz and Barnska, 2007), it was possible to assign some band features to specific compounds (table 7).

Sampling Methods: transmission methods

Transmission spectroscopy is the oldest and most straightforward IR method. The method is based upon the absorption of IR radiation at specific wavelengths as it passes through a sample. It is possible to analyze samples in liquid, solid, or gaseous form using this approach.

There are three general methods for examining solid samples in transmission IR spectroscopy: alkali halide disks, mulls, and films.

Reflectance Methods

Reflectance techniques may be used for samples that are difficult to analyze by the conventional transmittance methods. Attenuated total reflectance spectroscopy (ATR) utilizes the phenomenon of total internal reflection. A beam of radiation entering a crystal will undergo total internal reflection when the angle of incidence at the interface between the sample and crystal is greater than the critical angle. The critical angle is a function of the refractive indexes of the two surfaces. The beam penetrates a fraction of a wavelength beyond the reflecting surface and when a material that selectively absorbs radiation is in close contact with the reflecting surface, the beam loses energy at the wavelength where the material absorbs. The resultant attenuated radiation is measured and plotted as a function of wavelength by the spectrometer and gives rise to the absorption spectral characteristics of the sample.

The crystals used in ATR cells are made from materials that have low solubility in water and are of a very high refractive index. Such materials include zinc selenide (ZnSe), germanium (Ge), and thallium / iodide (TlI). Owing to the solid nature of the samples of resins, and the equipment present in the laboratory, this research work was performed in transmission mode (cf Materials and Methods section).

Results and discussion

After the collection of FTIR spectra of certified botanical resins, some spectral band positions were chosen owing to its contribution to differentiate spectra from different species (figures 35 and 36). This data was then used into chemometric analysis. Sample distribution patterns were investigated with principal component analysis (PCA). Score graphics using the first two components, revealed a sample agglomeration with good differentiation in 5 out of the 6 species. This method was validated by LDA with a 95,2 % of positive recognition. The aim was to establish a spectral databank in order to identify the botanical provenance of fresh resins. The first principal and the second principal components were enough to display the data structure, since they explained 79% of the total variance. Examining the score plot in the area defined by the first two principal components, a separation of the samples into five groups was found according to the different botanical species, except for B.stenophylla that overlapped the zone comprised by B. bipinnata. It is important to note that their distribution into the hyperspace is not the same: while B. bipinnata forms a single compact group, B.

A) B) C) D)
Stenophylla forms 2 groups one compact and the other overlapping the B. Bipinnata region.

These data are consistent with botanical findings, as it has been said on section two of chapter two, there are uncertainties that persist into the taxonomical classification of these two species: some authors consider B. stenophylla as a variation of B. bipinnata. As can be seen in the loading plot of the first two principal components, the bands f, e c and b corresponding to deformation of CH 3 ; CH 2 ; tension of C=O and tension of alkans CH 3 respectively characterized the first principal component.

Nevertheless f, e and c were placed close in the graph, meaning that these variables have almost the same information, therefore f and e columns could be removed from de data matrix without a big decrease of discrimination capability.

The result of doing so is shown on figure 40, where a greater dispersion of the data can be appreciated, although classes can still be recognized. Band g (C-O-C tension) was the variable that was placed closest to the center of the plot, therefore its impact on groups separation, is minimal in compared to the other bands in the model.

Linear discriminant analysis

Since the data structure analysis gave a good sample characterization, a classification model was built. LDA analysis was applied in order to find a predictive classification model, able to separate the 6 botanical species. As can be seen in the confusion matrix (table 9), LDA applied to the complete data set gave a recognition percentage of 95.2%, while only two resin samples were not correctly classified in the validation procedure. Complete statistical results can be consulted in annex 2.

3.9

Application to a botanical origin certified sample.

As there are over 80 species that produce resin in Mexico, we decided to perform a test with a resin with botanical certified origin, different from the six species that were included in the

Application to two commercial samples of copal.

The next step into the test of our model was to analyze a commercial sample as it can be seen on figure 42 for MEXJ1 and graph 43 for IZUM1, when PCA analysis was performed with FTIR data from these samples, both were placed into the hyperspace corresponding to both B. bipinnata and B. stenophylla species.

It is important to remember that MEXJ1 is a sample of yellow resin bought in a market from Mexico City, while IZUM1 is a sample of wood impregnated with a translucent resin that was bought at a seasonal market at Izúcar de Matamoros. Both cities are apart 200 km, confirming that even on fresh resins assignment of a botanical origin based in physical parameters is not possible.

Given the obtained results, a deconvolution of the area of FTIR spectra between 1200 and

spectra. Application of the technique to two commercial resins

One of the inconvenient that may arise when working with spectra of complex mixtures is the overlapping of the bands that different wavelengths, this phenomena is caused by the absorption of the radiation, at close frequencies, by different molecular bonds (of the radiated molecules) that vibrate to those same frequencies, or the same type of bonds placed in different environments within a same molecule. This phenomenon causes an increase of the amplitude of the signal in the spectra of the bond producing massive bands.

In the next stage of the research, we propose the decomposition of the massive signal from FTIR comprised between 1200 and 1300 cm -1 , to differentiate two close botanical species. This region was chosen because there were found more differences between B. bipinnata and B. stenophylla species.

As can be seen on figure 44, as a result of the differences in chemical composition of resins, differences in the number of signals arisen in some regions of the FTIR spectra can be appreciated. This feature allows the differentiation between botanical origins of fresh samples. Therefore, taking into consideration that production of secondary metabolites and, specifically triterpenes from resins, are strictly gene regulated, molecular composition can be used as indicator for the producing tree species. Given the genetic distance of the repertoired species of Bursera known at this day is highly possible that the botanical origin of archaeological Aztec samples is comprised between B.stenophylla and B. bipinnata species. To probe this conclusion, additional studies using chromatographic techniques were performed (refer to chapters four and five of this work).

Partial analysis of the surface of the samples

Considering that the surface of the copal is the part that is more exposed to degradation factors, but also the more accessible when sampling patrimony objects, a study of the variations of FTIR spectra of this kind of materials was performed.

Previous studies (De la Cruz-Cañízares, 2005) Light basically increases the initiation of the radical chain reactions, and oxidation proceeds mainly by light independent pathways [START_REF] Dietemann | Towards more stable natural resin varnishes for paintings[END_REF]. Nevertheless some differences have been remarked regarding the composition of resins under and without light exposition by other authors ( [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] Dietemann | Towards more stable natural resin varnishes for paintings[END_REF].

For this reason the most degraded surface found among the archaeological resins that we disposed (TMT), and two commercial samples (Huitzuco and SONB4) aged under natural light, were studied.

Comparison of partial analysis from commercial samples and a naturally aged sample of them

Aging of a sample of Huitzuco and a sample of SONB4 were conducted as follows: a mass of 30 g of each commercial copal was exposed to natural light behind a window facing south, during two months and a half (September-December), in Avignon, France. The material subjected to FTIR analysis was taken from the surface of the sample in a deep not exceeding 3 mm, as UV does not penetrate to deeper layers.

A comparative of the FTIR spectra of fresh and aged samples is shown on figures 49 and 50. As expected considering the changes occurring when ageing, the spectra of both intentionally aged samples developed much increased absorption in areas correlated to hydroxy groups (3450cm - 1 ), carboxylic acid groups (3200-2500cm -1 ) and carbonyl groups (1700cm 1 ) absorption . Together with the broadening of the bands a shifting in transmittance is observed, in order to asses the impact of this shifting of bands when using a FTIR-PCA model for the prediction of the botanical origin of a sample, test were performed using both data from fresh and aged samples. The results can be seen of figures 50 and 51. For samples both fresh and aged the botanical origin remained between the B. bipinnata and B. stenophylla regions.

Spectra variation between external and internal part of an archaeological sample TMT

An equivalent study to that one performed on light aged samples was conducted on an archaeological sample. Samples from the outer (more degraded) and the inner part of the fragment were collected. Separated spectra from these samples were collected and are shown on figure 53. Again a broadening in the bands of hydroxy groups (3450cm -1 ), carboxylic acid groups (3200-2500cm -1 ) and carbonyl groups (1700cm -1 ) can be appreciated in the spectra of the surface sample, changes in spectra certainly seem to be more dramatic, which is natural considering the age of the sample (around 500 years). Concerning the sample taken from the surface of the sample, it falls into the zone comprised between B. bipinnata and B. stenophylla species, and therefore from the data obtained from the surface the correlation to one of these two botanical origins can still be made. This results certified that ageing had an impact in spectra nevertheless even in the cases of very degraded archaeological samples, spectra still retain a good amount of information an can be useful when samples of the inner part of a piece are unavailable or when chromatographic methods can not be used.

Conclusions

Usually the sensitivity of vibrational methods is considered lower in comparison to other analytical techniques but in some cases, also components occurring in low concentration can be successfully analyzed. In this chapter we prove that global molecular profiling with FTIR can be successfully applied in combination to powerful chemometrics tools like PCA and LDA to validate and analyze complex data from spectra, to be used into the classification of resinous material from unknown origin.

The homogeneity of modern resins of certified origin made their analysis easier, and permitted to establish a spectral bank of data for these species.

The genetic closeness of B. stenophyla and B. bipinnata, was confirmed indirectly by the closeness of their resin composition.

In this chapter the deconvolution of the bands of the region between 1200 and 1300 cm -1 was performed onto fresh resins spectra, for the discrimination of samples from B. stenophylla and B. bipinnata. This method probed to be of great assistance when it comes to the classification of resins from close botanical origin.

It is important to note that conditions under which a sample aged significantly contribute to its chemical composition and consequently to its characteristic spectrum. This phenomenon was confirmed at different stages of this research, firstly when FTIR-PCA model was applied to the classification of archaeological resins as some of them fall in slightly different regions and secondly when partial analysis of the surface of aged resins was conducted, when partial spectra of fresh and more deteriorated resins were compared.

Auto-oxidation, condensation phenomena and degradation of triterpenoids can be cited among the processes that occur along time [START_REF] Dietemann | Towards more stable natural resin varnishes for paintings[END_REF]. Along ageing, hardness and consistency of a resin change, due to the evaporation of the volatile fraction [START_REF] Peris-Vicente | Estudio analítico de materiales empleados en barnices, aglutinantes y consolidantes en obras de arte mediante métodos cromatográficos y espectrométricos[END_REF].

From these considerations caution should be kept when applying a discrimination model based on FTIR data of fresh resins into archaeological samples, but still reliable information can be obtained from this method. Additional information coming from archaeological reports may contribute with invaluable information about these materials.

To sum up this method seems to be a fast tool that can help to reduce possible botanical origins when great diversity of botanical sources is found.

As it has been stated before considering their strong gene regulation on the secondary metabolites as triterpenes, and considering genetic distances among the 80 species of Bursera This came as consequence of their ability of separating organic compounds, from complex mixtures (Domenech, 2009). Chromatography comprises a group of methods for the separating molecular mixtures that depend on the differential affinities of the solutes between two immiscible phases.

In the last years, high performance liquid chromatography (HPLC), and gas chromatography (GC) coupled to a mass detector (MS), have progressively replaced other less sophisticated techniques such as paper chromatography or thin layer chromatography because they can reach a higher discrimination of the type of the materials present in artistic objects. These techniques can also provide a more complete description of the alteration products present in the sample. In this aspect their superiority compared to spectroscopic techniques is undeniable [START_REF] Doménech-Carbó | Study of ageing of ketone resins used as picture varnishes by pyrolysis-silylation-gas chromatography-mass spectrometry[END_REF].

Their major drawbacks are that they are that depending on the type of detection they may be destructive, and cannot document the organization of heterogeneous samples (Daher, 2010).

They also implicate pretreatments, like purification, extraction and derivatization that may be time consuming and modify original composition.

High Performance Liquid Chromatography (HPLC)

HPLC is a form of liquid chromatography used to separate compounds that are dissolved in solution. Separation is based on the partition coefficient of a given analyte. Partition refers to the relative solubility of the analyte between two liquid phases: a mobile one and a static one. There are two operation modes: Normal phase with polar stationary phase and non-polar solvent and reverse phase with a non-polar stationary phase and a polar solvent.

A classical HPLC device is constituted by a gradient controller, a pump, an injector, a column and a detector. A schematic view of their arrangement is presented on figure 55.

ANALYTICAL STUDY OF THE RESINS AND DISCUSSION ANALYTICAL STUDY OF THE RESINS AND DISCUSSION

PAOLA LUCERO

91

In this study the amount of resin needed for the analysis was directly correlated to the amount of gum present in the sample: at higher amount of gum, less terpenoids and more needed sample for the analysis. This study was performed by solving a known amount of rough resin in diethyl ether. After sonication and centrifugation, the residue left in the bottom of the solution was dried and weight. The percentage of gum was calculated from this data.

Finally, water was added to ensure the polarity of the residue. This data is consistent with, FTIR spectra. As presented in chapter 3, the samples from these species (B. simaruba and B.

grandifolia) afforded a broaden band in 3430 cm -1 . Generally speaking gum ratio is correlated to the producing species but this fact it cannot constitute a reliable factor for botanical identification, as gums are water soluble, widely distributed, and easy degradable materials. The big proportion of gum in B. grandifolia exudates prevented its study by HPLC, in fact some attempts were done but we concluded that for this kind of study, a much larger quantity than the actual amount of sample from what we dispose would be needed.

We performed the same type of analysis into archeological samples, solving a known amount of resin in ether; the ratios on non soluble matter founded are here reported (table 12). Nevertheless further test showed that this material was also insoluble in polar solvents. Therefore we believe that the percentage of insoluble matter could be constituted in a good amount by particular matter, possibly from mineral origin as. Owing to its low quantity it was not possible to determinate the nature of this material by this or any other analytical technique in our hands FTIR, and FTIR-ATR included.

To sum up, the ratio of gum present in archeological samples is almost zero, 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.

The second difficulty of analyzing these materials by HPLC relies in the nature of the resins themselves, which are chemically very complex: Witte et al. described the analysis of terpenoids like problematic and indicates that their complete chromatographic separation is almost impossible, even by using sufficiently long chromatographic colons, some of these compounds may not be totally resolved (Witte, 1986). Indeed, Bursera resins consist of compounds with very similar structure, and some of them may present identical times of retention. This complexity increases further with degradation process. The identification of composed on the basis of their time of retention or their order of elution is consequently not always reliable (Burger, 2003).

For the HPLC study, both standards and global resins were solved on methanol, the designed protocol can be consulted in the materials and methods section of this work.

Standard molecules

Liquid chromatography was applied to the study of six standard molecules (table 13), at 210 nm, which is the general wavelength number for the study of resins.

The availability of triterpenoic standards, and other materials often found in artworks like extended the application of chromatography to their identification, and the present research take advantage of this fact. In contrast the difficulty of obtaining standards of the oxidised alteration product formed during ageing from terpenoid compounds makes difficult the application of this technique to the identification of ancient materials. Therefore a GC-MS study was performed afterwards on resin samples to obtain a better characterization of concerned materials.

N° Molecule

Retention time (min) Molecules αamyrin, β-amyrin, lupeol, and lupenone were purchased from Extrasynthèse (Genay, France), αamyrone 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 samples.

Quantification of triterpenoids: analytical calibration

Standard solutions containing the above triterpenic compounds, at different concentrations were prepared and then injected by triplicate into the HPLC system. The outflow from the column was monitored by a diode array detector, operating at 210 nm.

ANALYTICAL STUDY OF THE RESINS AND DISCUSSION ANALYTICAL STUDY OF THE RESINS AND DISCUSSION

PAOLA LUCERO

94

Then the concentration of each of these molecules was calculated in each sample by introducing the peak-area into the equation of the curve. The linear calibration ranges, regression equations and, detection limits of these standard compounds were calculated. The results are listed in table 14. The linearity was tested in a variable concentration range according each molecule; their correlation coefficients are from 0.9989 to 0.9996. The limit of detection (LOD S/N = 3/1) and the limit of quantification (LOQ S/N = 10/1) were respectively lower than 3.7 µg/mL and 20 µg/mL.

Peaks were identified by co-injection with standard molecules, then corresponding area was taken into calibration equation and concentration of the concerned molecule was calculated. The results of these calculations are presented in annex 3 (tables 45-48).

Global analysis of certified resin samples

Chromatograms in liquid phase allowed a very clear interspecies distinction.

It is noteworthy to say that two regions of peaks can be distinguished on each chromatogram: the first one going from 0 to 24 minutes and the second running from 24 to 35 minutes.

The first zone corresponds with components from lower molecular weight and highest polarity (compounds with a molecular mass that corresponds to mono and sesquiterpens in UPLC-MS).

The second group of peaks in the chromatogram corresponds to molecules of higher molecular weight and lower polarity; these molecules were identified by co-injection with standard molecules as triterpenes.

ANALYTICAL STUDY OF THE RESINS AND DISCUSSION ANALYTICAL STUDY OF THE RESINS AND DISCUSSION

PAOLA LUCERO

95

In one hand, the composition of the more polar fraction is very different in both quantitative and qualitative terms for both species.

In the other hand a very homogeneous composition of the triterpenic fraction in qualitative terms was found for all resins (botanically certified, archeological and commercial samples)

nevertheless the relative amount of each compound, for each species is quite particular.

These facts allowed us to model a molecular fingerprint by means of PCA this subject will be further discussed in this chapter.

In figure 56 we present a chromatogram of B. bipinnata where the two above mentioned areas of peaks are distinguished: in red the polar one, and in blue the tritrepenic one. All triterpenes, identified in this research were assigned a number, according to their elution order. Number assignment will be kept all along this chapter for the identification of these molecules in other chromatograms.

Comparison of global chromatograms from certified origin resins

Ad it was said before, differences can be remarked concerning the global molecular composition of each species. In one hand table 16 resume the presence and absence compounds of lower molecular weight and more polarity (from the red zone), in the other hand table 17 resumes the presence and absence of triterpenoids (blue zone) in each species of resins.

Concerning the second part of the chromatograms it is worthy to say that other triterpenic compounds were present in each sample, nevertheless it is at this point that UV/Vis reach is limits concerning differentiation of molecules with close structure, and therefore a more sensitive method as GC-MS was used for the detection and identification of these molecules, for an account into this matter refer to the GC-MS chapter of this work. Regarding the first part of the chromatogram we chose a notation for compounds beginning either with letters A or B.

B.bipinnata - + + + - - - + + - + - B. excelsa - + - + + - - - + + - + B. laxiflora + + + + + + - - - - - + B.laxiflora - - + + + + - - - - - + B. penicillata - - - + + - + + + - - - B. simaruba - + + + - - - - - + - - B. stenophylla + + + + - - - + - - - -
Compounds with an "A" notation are widely distributed in the 6 species, most of the time A1 is a major peak in their chromatograms. While "B" compounds are specific for each species, meaning that their presence and quantity varies a lot among species. 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 bipinnata (cf figure 58). Concerning the polar zone B. stenophylla seems to be richer qualitatively speaking, as at least 4 major peaks can be distinguished in this species. For B.

B. stenophylla B. bipinnata

bipinnata only two major peaks can be seen, in the polar zone. Concerning B. excelsa two lots can be distinguished as well, based in the molecular composition of both more polar and triterpenic fractions, these differences can be appreciated on figure 60.

Although three major peaks can be distinguished in the polar zone, based upon their retention times, different identifications can be attributed to each one. Concerning the triterpenic fraction, type 1 seems to have a wider variety of different molecules than type 2. Identification of triterpenic compounds is as follows: 1) 3-epi-lupeol, 2) lupeol. Similarly in the case of B. simaruba: two molecular profiles based upon the composition of the resin can be described: a first one which contains three major peaks in the more polar zone (A3, A4 and B4) of the chromatogram, while the second one has a single major peak (A1) in the same chromatographic region (figure 61). Concerning the triterpenic fraction of the resin, both profiles have an identical composition. Finally the species that exhibits the greater diversity in molecular composition is B. laxiflora, within this species six groups could be distinguished and two of these groups (lots 1 and 2) counted more than five individuals (cf figure 62).

B. excelsa, type 1 sample 27a2

B. simaruba, lot 1 sample 70a1Q

B. laxiflora

6 Comparison of global chromatograms from commercial resins

Concerning commercial copals with a whitish to yellow aspect two species were certainly differentiated by HPLC : B. bipinnata and B. steophylla. An important remark to these results is that no correlation can be established between botanical origin and physical aspect (color) of a resin, confirming data from the literature (Régert, 2008) From this point of view, commercial samples ATZB1 and Huitzuco are likely to be B bipinnata resins, as the molecular composition determined by HPLC-UV/Vis means, seems to be the same.

Here in figure 63, we present some chromatograms of these samples. In the other hand based upon the great variation observed among the molecular profile of the studied certified resins, it is highly possible that commercial resins CHOB1, CHOB3, CHOM1, Identification of triterpenic compounds is as follows: 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

Many commercial samples, afforded chromatograms with too few or no triterpenes at all (figure 65), The physical aspect of this copals was white in the case of: MEXM1, ATZB2, Colima L2, OAXC1, OAXC2 and OAXC3, yellow for Incienso ATZJ2, and red for IZUR1. This information is a sign of a very different botanical origin, for these resins.

In many cases, most of their molecular compounds are located in the intermediate part of the chromatogram. Further studies need to be done upon these resins in order to find their botanical origin, again a more sensitive technique as GC-MS Some of these resins were bought within the same geographical location and nevertheless have a very different botanical origin. This has an impact towards the utilisation of commercial resins into restoration works: all commercial resins should be tested in order to ensure that its 104 composition is as closer to original material as possible, to avoid possible interactions, and accelerated degradation from a more complex mixture of compounds [START_REF] Dietemann | Towards more stable natural resin varnishes for paintings[END_REF].

ATZJ2

Comparison of global chromatograms of archaeological resins

The purpose of the study of the archeological resin samples was to offer a methodology that can corroborate some assumptions about the use of copal in pre-Columbian times as adhesive, and molding material.

Despite the age of archeological samples, and their preservation under different conditions of relative humidity, pH, and temperature, their molecular composition retained most of the triterpenic fraction compounds except for 3-epi-ß amyrin and T3.

Considering the chromatogram profiles of Aztec materials coming from Templo Mayor, and those from botanical certified samples, HPLC results may suggests as botanical origin for these samples either B. bipinnata or B. stenophylla. In order to corroborate these results PCA was coupled to HPLC data, GC-MS was applied as well, and results of this analysis will be presented below and in chapter V.

The main molecular difference between these two species is compound T3, unfortunately it tends to disappear from aged resins.

In the molecular level, the study of archeological resins by this technique allowed us to detect molecules that were not present in fresh samples, the peaks corresponding to these molecules can be appreciated from minutes 32.5 to 36, in figure 66. Here we present the chromatogram of sample 173, the triterpenes present in fresh samples are marked in black, while C1 to C4 in green, represent the molecules either from higher molecular weight or higher polarity than molecules present in fresh samples. An adapted protocol for the study of these samples can be found in the materials and methods section of this work.

We defined this profile for archeological samples as profile A.

Fig 66 Chromatogram of archeological sample with triterpenes and ageing related products (C1-C4).

Identification of triterpenic compounds is as follows: 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. It is important to note that these molecules are not always present in archeological materials; they are absent in TMT and 52 (figure 68) these profile was defined as profile "B". From these results as well as from the solubility test we conclude that no other organic material enters in a great proportion in the composition of these samples. Concerning inorganic materials a small amount of a particle matter maybe insoluble in both polar and apolar solvents was found as it was said on section 4.1 of this chapter.

The results of this experiments yield important information for archeologist that were previously unavailable. Firstly this work confirms that a botanical origin of copal among the great variety of over 80 possible species was preferred by the Aztec priest. This origin is the same either for molded figurines, offerings to the Gods or used as adhesives for ceremonial knives.

In our days botanical distinction between B. stenophylla and B. bipinnata is unclear only with very sophisticated techniques slight differences in molecular compositions of their resins can be detected.

Concerning the botanical origin of Maya sample from Chichén Itzá (figure 69) we can hypothesize a very different botanical origin from the samples coming from Templo Mayor.

Firstly the qualitative content of its triterpenic fraction is quite different quantitatevily from the resins that we studied before, even more the peak area for the α amyrin/ β amyrone mixture is the major peak in the triterpenes zone, while in the rest of the archaeological samples, it was the 3-epi-lupeol peak which has the greatest area.

Its triterpen composition may correspond to a Burseraceae genus origin, but we can definitively discard a B. simaruba origin, that was related to Bonampak murals in Yucatán region (Magaloni,

HPLC coupled to PCA analysis for botanical certified resins

As seen on the previous sections natural terpenoid resins have been identified by HPLC, coupled with PDA detectors in certified, commercial and archeological resins of Bursera. In order to compare the performance of FTIR and HPLC discrimination power, PCA was applied to HPLC data.

For this part of the study, both volatile (table 16) and triterpenic (table 17) compounds were selected in chromatograms, according to their retention times, peak areas were used in a indirect way: considering that the analysis was performed on different amounts of crude resin (and therefore different concentrations) according the species of resin, the percentage of relative area was chosen for PCA analysis, to build the discrimination model.

B. simaruba was excluded from PCA analysis because lupeol and epi-β-amyrin were never resolved into different peaks, the same happen to 3-epi α-amyrin and β -amyrin in this species.

A data matrix with 40 rows (resin of certified origin samples) and 22 columns (variables, in this case relative area of the peaks in the chromatogram) was built (annex 3, tables 41, 42 and 43).

In figure 70 the structure of the data, using the first two principal components, is displayed.

bipinnata, B. penicillata

In this case the first two principal components were enough to display the data structure, and they explained 46.5% of the total variance. Examining the score plot in the area defined by the first two principal components, good discrimination results were achieved, even more B. bipinnata and B. stenophylla were correctly discriminated also B. penicillata is 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.

When analyzing loading plot (figure 71) the molecules that exhibit the highest influence onto first component (in green) in the positive side are triterpenes: lupenone, 3-epi-α-amyrin,

ANALYTICAL STUDY OF THE RESINS AND DISCUSSION ANALYTICAL STUDY OF THE RESINS AND DISCUSSION

PAOLA LUCERO 110 lupenone, α-amyrin/ β-amyrone. In negative side with the highest impact B8 and B1 from the more polar fraction have the highest discrimination power.

As can be seen in the loading plot B2, B5, A3 and and A4, all volatile compounds show to have the higher impact on second component together with 3-epi-lupeol of the triterpenic fraction.

Generally speaking all triterpenes play a major role into discrimination except for lupeol which seems to have little impact on both components, together with A1.

On the second component B3 and B5 have the higher impact in discrimination, they both seem to content 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.

Purification by Solid Phase Extraction (SPE)

Sample preparation is necessary when molecules that interfere with the determination of the analyte are present within a matrix. To eliminate those interferences a number of techniques may be applied, namely: filtration, liquid-liquid and solid-solid extraction, etc.

In the last years solid-solid extraction has evolved a lot, not only allowing the purification but also the concentration of the target(s) molecule(s) within a sample.

General Methodology

For SPE cartridges, columns and plaques filled up with adsorbents are used. In the present work a reverse phase Chromabond C-18ec phase was chosen. Adsorbent was chosen considering its good affinity to triterpen molecules and its low affinity to other compounds. The volume of the cartouche was defined by the volume of the sample and the quantity of analyte present in the rough resin. In the present case for the analysis of commercial resins a 6 mL cartridges was selected, while a 1 mL was chosen for archaeological samples.

Generally speaking every SPE protocol has at least 4 steps:

1) Conditioning. This stage allows the elimination of pollutants from the adsorbent at the same time that allows its activation. For this stage 6 mL of methanol were used. It is important to not let run the column dry.

2) Deposit of the sample. This step consists in the deposit of the sample onto adsorbent surface. To ensure better purification efficiency the velocity of filtration should be controlled. Experimental data show that for cartridges with 1 mL volume an adsorbent granulometry of 50µm a optimal time of deposit is 0.7 to 1 mL/ minute, for 6ml volume cartridges of 5 -6 mL are recommended.

3) Washing of the adsorbent. It is at this stage that the impurities without any affinity with the adsorbent will be eliminated from the solid phase. For this purpose a solvent or a mixture of solvents with good affinity for impurities and poor affinity to the analyte, should be used.

CHAPTER IV

CHROMATOGRAPHIC TECHNIQUES: HPLC UV/VIS

ANALYTICAL STUDY OF THE RESINS AND DISCUSSION ANALYTICAL STUDY OF THE RESINS AND DISCUSSION

PAOLA LUCERO 116 4) Elution. At this stage a solvent with the maximum affinity to the analyte must be used, to recover it. Its volume must be low as it will provide a higher pre-concentration. An adsorbent with lower particle diameter will afford lower elution volumes.

The "lit volume" is the amount of solvent used for conditioning, rinsing and elution and is about two to three times the volume of adsorbent

In the present work for the study of commercial samples the column was conditioned prior to use by drawing 2 times 3 mL of methanol followed by the same volume of distillated water. The prepared sample (2 mg/mL) was loaded onto and drawn through the column. The column was then washed with 3 mL of a mixture methanol-water (5:95, v/v). The cover of the manifold was then removed and the cover was wiped with a tissue, to remove drops of washing solution.

Triterpenoids were then eluted from the column with 2 x 2 mL of methanol. The eluate collected was then analysed by means of HPLC.

The result of applying this technique are shown on figure 74 as it can be see on these chromatograms SPE show that it effectively separated the compounds corresponding to A) polar compounds and B) apolar compounds .

Polar fraction Triterpenic fraction Fig 74 Chromatograms of A) the washing product and B) the elution product. Identification of triterpenic

compounds is as follows: 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

The extraction method described here for the qualitative analysis of triterpenoids in the matrix of a natural resin proved to be useful in terms of simplicity and general applicability. It should prove to be valuable in further studies in natural resins from Mexican Bursera origin and generally speaking with a triterpenic composition.

Conclusion

The usefulness of this technique is undeniable concerning molecular profiling, for differentiation between resins with different botanical origin. HPLC data coupled to PCA was able to discriminate botanical origin of fresh resins from very close species as B. bipinnata and B.

stenophylla, which were hard to differentiate with FTIR-PCA.

The performance of the mathematical model constructed with HPLC data in PCA and validated by LDA exhibit a high performance with a100% recognition in the fitting matrix and a 95% of positive recognition in the cross validation matrix.

The study of chromatograms from commercial resins show as well very interesting results: from these samples, two were identified as B. bipinnata resins, 9 as B. stenophylla resins, nine afforded chromatograms with too few or no triterpenes at all.

From the B. bipinnata both of them were white, concerning the B. stenophylla resins, one was white, one was red and one was a "myrrhe".

From the 9 resins from unknown origin 2 were yellow, one was red and the rest were white.

Confirming that no correlation can be established between the physical aspect and presentation of resins and their botanical origin.

Concerning ageing, liquid chromatography allow us to confirm that Bursera resins seem to undergo significant changes along time: from one side the evaporation of the volatile fraction and in the other one the oxidation and maybe the polymerization of the triterpenic fraction.

These modifications can be assessed only partially by means of HPLC-UV/Vis, as it was show in the chromatograms from archaeological samples and therefore a deepened study should comprise the isolation of compounds C1 to C4 and its formal study by R.M.N.

Interestingly these changes nevertheless are not always present in archaeological resins, and therefore an insight into the mechanism of their formation and maybe a correlation between ageing conditions and their presence should be done.

Finally only few chemical species seem to be stable during natural aging: A4 of the non triterpenic fraction at retention time of nine min and all the triterpenic molecules except from 3epi-α-amyrin, which nevertheless exhibited a tendency towards a changing on its proportions.

Gas Chromatography-Mass Spectrometry

Gas Chromatography (GC) is a technique described, for the first time, by Martin and James in 1952. It is based upon the partition of the analytes between two phases: an inert, not very viscous, highly pure carrier gas as mobile phase and a liquid or solid stationary phase [START_REF] Bartle | History of gas chromatography[END_REF]. The use of this technique alone is limited when facing complex mixtures of compounds (Domenech-Carbó, 2008). This drawback can be resolved by coupling this technique to gas chromatography (GC-MS).

In a Mass Spectrometer (MS), the identification of an analyte is achieved by the ionization of a molecule, its fragmentation into smaller ions and their separation on the basis of the mass/charge ratio by an analyzer. This may be a quadrupole, an ion trap or a magnetic sector. In the present work an ion trap was used.This technique yields information about the molecular mass of a compound, its global formula and from the fragmentation pattern, about its molecular structure.

When analyzing complex mixtures it is possible to draw qualitative and quantitative information [START_REF] Colombini | Chemical study of triterpenoid resinous materials in archaeological findings by means of direct exposure electron ionisation mass spectrometry and gas chromatography/mass spectrometry Rapid[END_REF]. Mass spectrometry is therefore a powerful tool for analysis involved in the identification of organic materials from artworks, as it has the potential to resolve molecular structures. Typical equipment for modern GC-MS consists of a cylinder containing the carrier gas -Helium, in this work-, an injector, a capillary column inside a temperature regulated oven, an interface (by which most of the carrier gas is depleted), a mass detector and the electronics (figure 75).

More detailed information about the technique and its theory can be found elsewhere [START_REF] Hubschmann | Handbook of GC/MS[END_REF].

Derivatization

It is a preliminary chemical treatment which main goals are to make compounds more volatile or less polar and consequently suitable for GC analysis, to lessen absorption phenomena into column due to functions alcohol and acid type, to stabilize thermally substances analyzed and to contribute to a better separation of compounds from similar structure [START_REF] Hovanessian | Différenciation de substances naturelles par diverses techniques analytiques: spectroscopie IRTF, CLHP / uv-visible / fluorimétrie et CPG /SM[END_REF]. The used of derivatization process is reported in the materials and methods section of this work.

Trimethylsilylation of carboxylic and hydroxyl groups was achieved by a method previously described by Mathe et al. in In the present research, this technique was firstly used for the analysis of freshly collected resins from 6 species. Then triterpenoid composition was compared to that of selected commercial and of archaeological samples.

The study of standard triterpenoic molecules

As in the HPLC study of this work the same 9 standard molecules were studied; they presented ursane, oleanane and lupane skeletons and their TMS structure is presented on table 20. The obtained mass spectra of standard compounds were stored for the construction of a bank of mass spectra adapted to the study of triterpenes. Later, the comparison between the mass spectra of the chromatographic peaks obtained when analyzing commercial, archaeological and botanically certified samples, allowed the identification of the compounds present in each sample.

Ursane Oleanane Lupane

According to earlier studies of our research team (Mathe, 2004)

Insight on triterpenic composition for samples of certificated origin

In this section we will present an insight into the identification of standard molecules in the triterpenic fractions of botanical certified resins. For this purpose a comment on chromatograms obtained under the protocol 2 conditions will be addressed.

In figure 81 the entire chromatogram of triterpenic of B. bipinnata is presented. As it can be seen only minor peaks are detected before 30 min, even more spectra from these peaks are related to secondary products of TMS reactions. Therefore for a better identification of compounds a partial TIC chromatogram between minutes 30 and 40 using gradient two, are presented in this section.

As it has been said complete account on identified triterpenoids and their retention time for each botanical studied species can be found in annex 4 (table 49).

B. excelsa triterpenic composition

Although only two out of the nine standard molecules were present: (I) 3-epi-α-amyrin, and (III)

3-epi-lupeol this sample is not as poor in triterpenes as one might suppose. In addition to these molecules other five triterpenes were detected. One of these markers was detected in B. laxiflora sample as well, in (figure 83) it is designed as EL1 (E for excelsa, L for laxiflora plus a number).

B. grandifolia triterpenic composition

The GC-MS results allow us to confirm the triterpenic nature of this resin, and to have some insight on the compounds that are present in it. This is of great value considering that it was impossible to perform HPLC-UV/Vis analysis on this material.

In the resin from this species 4 compounds among the standard studied molecules were

B. laxiflora triterpenic composition

This was the species that confronted us to a more variable triterpenic profile in qualitative terms.

Therefore in this section are presented the two major molecular profiles encountered.

For the lot 1, seven out of the nine standard molecules were identified, the only absent compounds were two ursane squeleton molecules (VII) α-amyrine and (VI) α-amyrone (figure 85).

Additionally two other triterpenoid molecules were detected, identified as EL1 entering as well in B. excelsa composition and L1 exclusive from B. laxiflora species. In the second lot molecular composition the same seven standard molecules along with the two markers, were identified. The difference between the two lots relies in the compound detected between minutes 38 and 39 (figure 86).

B. penicillata triterpenic composition

This resin appears to be richer in triterpenoids than the other ones; in fact many important peaks were identified after 39 minutes. This is why we decided to present both chromatographic zones. Figure 88 presents a partial chromatogram that runs from minute 38 to minute 50. From this zone three other molecules were chosen as markers (P5 to P7). Therefore molecules P1 and P2

can be seen at the beginning of the chromatogram.

B. simaruba triterpenic composition

As it can be seen on figure 89, eight out of the nine standard molecules studied were present in this sample, the only absent compound was lupenone, in contrast five other triterpenic compounds were detected, three exclusive for B. simaruba (S1-S3).

Study of archaeological samples

As we expected archaeological samples are rich in triterpenic compounds, these compounds were detected from 39 min of the chromatogram using protocol 1. The molecular composition of Aztec samples (26,[START_REF] Carreon-Blaine | Reseña de las ofrendas del Templo Mayor de Tenochtiltlan de Leonardo Lopez-Lujan[END_REF]52,84,140,173 and TMT) can be correlated to that of B. bipinnata and B. stenophylla while the molecular composition of Chichen Itzá sample is completely different from that of Aztec and botanically certified resins, so we can conclude that its botanical origin is not within the studied species.

Molecular composition of archaeological sample 52

The global chromatogram of this sample is consistent with the former studied chromatograms. It shows no peaks before the 39 min.

The molecular composition of triterpenic fraction is close to that of samples 26 and 5: the nine standard triterpenic molecules were identified, plus the 4 markers for B. bipinnata and B.

stenophylla (BS1 to BS4) resins.

NB. Although β-amyrin (V) is not marked and identified in the corresponding section of the chromatogram (figure 93 A and, B), some minor quantities of this compound were detected.

A) B)

Molecular composition of archaeological sample 84

As it can be seen on figure 94A the molecular composition of this sample is complex, but its complexity is not related to the composition of the triterpenic fraction, as it is rather simple fraction.

Among triterpenes the nine studied standard molecules were detected, again some minor quantities of (V) β-amyrin were detected, along with the four BS molecular markers.

A) B)

Molecular composition of archaeological sample TMT

As it can be seen on figure 96 A) many minor peaks corresponding to non triterpenic compounds were detected between 7 and 38 min. In the triterpenic fraction the nine studied standard molecules were detected, three of the four BS molecular markers were found with the absence of BS4 (figure 96 B).

Molecular composition of archaeological sample 140

As it can be seen on figure 97A many peaks corresponding to non triterpenic compounds were detected between 6.7 and 23min.

As their fragmentation patterns are different their nature certainly differs a more detailed informations about them is beyond the scope of this work owing to time restriction.

In the triterpenic fraction of this sample the nine studied standard molecules were detected, two of the four BS molecular markers were found: BS1 and BS2.

Molecular composition of archaeological sample from Chichén Itzá

As it can be seen on figure 98 A, the molecular composition of this sample differs from that of the other archaeological samples, this is consistent with our findings by HPLC-Uv/Vis.

As the fragmentation patterns from the peaks detected between 10 and 27min are different one from another their nature certainly differs, a more detailed information about their molecular structure is beyond the scope of this work owing to time and equipment restrictions.

Also the triterpenic fraction of this sample differs greatly compared to that of the other archaeological samples: the relative proportion of 3-epi-α-amyrin, 3-epi-β-amyrin, and 3-epi- Concerning the proportions of β-amyrone and α-amyrin, they are considerably more important in this sample than in the other archaeological ones.

Finally two markers BS1 and BS2 were detected in this sample.

A) B) Fig 98 A) Complete chromatogram and B) zoom into triterpenic zone of archaeological sample Chichén Itzá

Identification of triterpenic compounds is as follows: The abundance of secondary products visible in GC-MS and the high number of unidentified peaks is not surprising if we consider that this material is in fact a fifteen century resin and the fact that oxidation phenomena are quite active in this kind of molecularly complex materials.

(I) 3-epi-β-amyrin α-OTMS, (II) 3-epi-α-amyrin α-OTMS, (

Study of commercial samples

In this section we will present the chromatograms of some selected commercial samples, the choice of studying them was determined in part by the results obtained by FTIR and HPLC-UV/Vis and because of their representatively considering the other commercial resins.

Molecular composition of Sidral

As can be seen on figure 99 A, the global chromatogram of Sidral exhibits an important amount of peaks between 13 and 30 min. The triterpenoid fraction of Sidral is identical to that of B. bipinnata and B. stenophylla, the nine identified standard molecules plus the four markers BS are present. Therefore its botanical origin is without doubt among these two species.

Molecular composition of commercial sample SONB4

According to the obtained GC chromatogram, this sample has a very similar molecular composition to that of Sidral with only minor difference: the absence of BS4 (figure 100).

Considering that this sample is a fresh sample and that BS4 was detected in many of the archaeological resins one might hypothesized that this compound was never part of the composition of this sample.

Comparison between molecular composition of ATZJ2 and SONB4

In figure 101 are presented the complete chromatograms of sample ATZJ2 and SONB4. Both samples were analyzed using the same gradient, and triterpenic zone of ATZJ2 is empty, not a single triterpenoid from the standard molecules was the chromatogram detected, neither any BS markers.

Molecular composition Oaxc2

The chromatogram showed on figure 102 confirms the absence of triterpenoids that we assumed from the study of the HPLC chromatogram for this sample. These samples correspond to two different kinds of copals a red one and a "piedra" one. The physical aspect of both samples differs a lot from white copals; nevertheless their molecular composition is close to that of SONB4.

As it can be seen on 103 A and B chromatograms, the nine standard triterpenoic compounds were identified on them, as well as the four molecular markers for B. bipinnata and B. stenophylla species.

These data strongly suggest that these two resins have a common origin and it can be correlated to the above mentioned species. Concerning the volatile fraction of these two samples differences can be inferred from their global chromatogram presented here in figure 104, SONR1 the sesquiterpens α-pinene and azulene, as well as limonene derivatives were detected. In IZUP1 α-pinene and myrtenol were detected. All the identifications were performed using NIST library 2011 version.

Considering the derivatization process that samples were subjected to, the complete characterization of volatile fraction is not possible, being light molecules some of them might have been volatilized.

Therefore an accurate procedure for the study of these molecules should be followed for their study. In this section we will report some results of comparing in one side the chromatograms of a fresh resins and the same resin aged under natural light from September to December 2011, and in the other side a chromatogram from the material obtained from the surface of TMT archaeological sample and the inner part of a piece of this resin sample.

Comparison between SONB4 fresh and naturally aged sample compositions

The ageing of the sample was conducted according to the procedure described below. A sample of 30 g of commercial copal was exposed to natural light behind a window facing south, during three months (September-December). The material subjected to GC-MS analysis was taken from the surface of this sample in a deep not exceeding 3 mm. As it can be seen on figure 105 A, the first impact of ageing in a copal is the lost of a big proportion of its non triterpenic compounds. Concerning the triterpenic fraction changes in proportion of triterpenes occur as well: (II) 3-epi-α-amyrin, (VII) α-amyrin show an augmentation in their proportions.

The same happens with BS3 and BS4. All these molecules are marked with an "A" from augmentation in the chromatogram (figure 106).

These findings are in accordance to those from De la [START_REF] De La Cruz-Canizares | Study of Burceraceae resins used in binding media and varnishes from artworks by gas cromatography-mass spectrometry and pyrolisis-gas chromatography-mass spectrometry[END_REF]. Her team analyzed both fresh and aged resins, and they found that ratio β-amyrin/α-amyrin peak area ratios were of around 0.41 in fresh resins while it droped to values between 0.30 and 0.15 in light and heat aged samples. Although ratios were not measured in the present study the results of our experiences ands those from De la Cruz-Cañízares suggest that β-amyrin is affected to a greater extent by heat and light than /α-amyrin.

Molecular composition variation between external and internal part of an archaeological sample TMT

The chromatograms of both the inner part and the surface of TMT are presented here, some differences can be remarked, as in the case of the intentionally aged sample, some peaks exhibit an augmentation into their proportions, this is the case for: (II) 3-epi-α-amyrin, (III) 3-epilupeol, (V) β-amyrin, BS1, BS2, BS3, BS4, but the most spectacular change is the one occurred in (VII) α-amyrin proportion that doubles its size. All these molecules are marked with an "A" from augmentation in the chromatogram (figure 107). As in the case of artificially aged samples α-amyrin seems to be then less affected by natural ageing. The ageing of archaeological samples occurred under a temperature that can be estimated around 20ºC , a no loght exposure.

The major difference between SONB4 and the surface of TMT resin is the detection of a compound at 39.78min, it is marked in the chromatogram as "N" and its mass spectra is presented in figure 108. Also it allowed the study of terpenoid fraction of B. grandifolia. These analyses are important because it allowed us to identify (I) 3-epi-β-amyrin, (II) 3-epi-α-amyrin, (IV) β-amyrone and

(VI) α-amyrone, and therefore to confirm the triterpenic nature of this resin.

Concerning the case of B. bipinnata and B. stenophylla, this technique allowed us to establish beyond any doubt, that the triterpenic fraction of both resins is identical. This fact explains the results obtained by FTIR and HPLC techniques.

Another great advantage of using GC-MS for the analysis of botanically certified resins was the possibility of detection of compounds that could be used as markers for each different botanical species. The structural characterization of these compounds by accurate techniques as NMR will certainly contribute to differentiate these resins in other commercial and archaeological samples.

In the case of Aztec resins, GC-MS permitted to correlate its origin to B. bipinnata and B.

stenophylla.

As the only difference in composition of these resins is found within the non-triterpenic compounds, the chemical distinction between these two origins in aged samples is impossible using chromatographic techniques.

Concerning Maya sample from Chichén Itzá, we could only confirm the results obtained by HPLC-UV/Vis: its composition differs from the molecular profile of all studied certified resins.

Its botanical origin certainly belongs to a species that was not studied in this research. Regarding the presence of BS1 and BS2 within this sample and considering the results of ageing experiences two conclusions can be assumed: the first one is considering this sample as a mixture of resins, which would not be surprising as [START_REF] Ryan | Chemistry and ethnobotany of commercial incense copals, copal blanco, copal oro, and copal negro, of North America[END_REF][START_REF] Van Gemert | Characterization of copal incense from Mesoamerica: Identification of residues by GC-MS[END_REF] documented anthropomorphically mixed resins in Maya culture. The second possibility for this Regarding the study of commercial samples it provides important information: the aspect, color and the place of acquisition of the resin have no correlation to its chemical composition, and therefore its botanical origin: a "piedra" copal bought in Puebla region (IZUP1), a white copal from Mexico City market of Sonora (SONB4) and a red copal (SONR1) also bought in Mexico

City exhibit a very close triterpenic molecular composition. The composition of the three commercial resins can be correlated to a botanical origin within B. bipinnata and B. stenophylla species.

As these samples were bought in more or less distant regions their botanical origin may reveal that the exploitation of this species along Mexican territory is more or less prevalent.

The botanical origin from some other commercial resins that were studied remains unknown; this is the case for a red copal bought in Oaxaca region (Oaxc2) and a yellow copal bought in Mexico City (ATZJ2). Its molecular composition from the fragmentation patterns of their molecules is neither triterpenic nor diterpenic.

The evaluation of changes induced by ageing shows in the short term that the non terpenic molecules are highly affected. In the long term triterpenic fraction is modified: the formation of an unknown compound that we designed as "N" was appreciated as well as changes in the proportions of many triterpenes, the most spectacular change was that in the proportion of αamyrin whose peak doubled its size in the case of the more degraded resin.

It is hoped that this research will stimulate further investigations towards a better characterization of the molecular composition of Mexican Bursera resins.

General Conclusion

In this research three types of samples of Mexican copals were studied: botanically certified archaeological samples and commercial. Here we present a recapitulative table of the botanical origin found for archaeological and commercial samples. Two analytical approaches were used in this study: a spectrometric one, based on FTIR and a separative one based on HPLC and GC-MS chromatography. Each approach had advantages and afforded valuable information.

From FTIR and HPLC data a maximum of information was extracted with the aid of modern statistical techniques as PCA and LDA. The use of multivariate statistical tools allowed building mathematical models for the classification and the prediction of the botanical origin of unknown samples of resin. According to LDA results the performance of the two models was evaluated and the value of positive recognition was of 95.2% for the model created with FTIR data, and 95% of correct recognition in the fitting matrix for the model created with HPLC-UV/Vis data.

These models were used for the identification not only of fresh commercial resins but also for archaeological resins. The changes induced by ageing into these samples that altered their composition do not seem to prevent the use of these models for their classification.

In the case of archaeological Aztec samples its relation to B. stenophylla or B. bipinnata species is clear, and confirmed by the results obtained with the three analytical tools. The uncertainty between these two possible origins arisen from the fact that tritrepenic composition of both resins is identical. Only some molecular differences occur on the essential oil and/or gum fractions, which tend to disappear from aged resins, this fact was confirmed by the results obtained by the chromatographic analytical techniques.

Concerning the Maya resin sample we were able to discard its membership to any of the studied resins. Nevertheless a doubt persists about its composition, as it may be constituted of a mixture of two or more resins. Further studies should be performed on resins from Bursera spp. endemic of Yucatan peninsula, then in order to clear out this point.

Although spectroscopic techniques are considered to be less sensitive than separative techniques, their hyphenation to multivariate statistical techniques seems to overcome its alleged limitations.

It was the case at least for this study were as we mentioned above the performance of the discrimination model slightly overcome the model created with HPLC-UV/Vis data in the validation matrix.

Furthermore this performance in discrimination was achieved using only eleven variables for FTIR (band positions in the spectra) versus twentytwo variables in HPLC (relative area of the peaks in the chromatograms).

Also FTIR has the advantage of being a fast, less expensive tool with a higher sensitivity as in our experiences only 2 to 3µg were needed, regardless of the nature of the sample (botanical origin, age).

When a greater sensitivity in the discrimination of two closely related species was needed the deconvolution of selected spectral zones seems to be a good option. In this study the bands between 1200 and 1300 cm -1 were deconvoluted allowing the discrimination of different botanical origins.

GENERAL CONCLUSION ANALYTICAL STUDY OF THE RESINS AND DISCUSSION ANALYTICAL STUDY OF THE RESINS AND DISCUSSION

PAOLA LUCERO

157 An advantage of chromatographic techniques over spectroscopic techniques is the fact that they can contribute with an overview of the molecular composition of the sample, from the chromatograms the number and the kind of compounds present in these samples was determinated. When standards were available the identification and even the concentration of some triterpenic compounds was calculated.

The HPLC-UV/Vis-PCA model was in fact the only of the three techniques able to discriminate between the origin of fresh samples of B. bipinnata and B. stenophylla resins. The advantage of constructing a model considering both triterpenic and non triterpenic compounds increases the sensibility of the model in this aspect.

HPLC-UV/Vis also was the only analytical tool that documented the presence of molecules that could correspond to saponines or to degradation compounds in archeological samples.

Finally GC-MS was the only tool from what information on particular compounds present in the resins could be obtained.

Its sensibility towards the detection of different triterpenoids exceeds largely the performance of HPLC-UV/Vis, as the limitations of UV/Vis capabilities into the discrimination of closely related structures are well known.

The characterization of botanically certified resins by GC-MS allowed us to find molecular markers for each botanical originated species, that hopefully will be useful in the future for the study of commercial and archaeological resins.

Finally the results of the characterization of commercial samples revealed that the origins of some commercialized resins in Mexico are B. bipinnata and B. stenophylla species regardless of the appearance and the presentation of the copals.

In the other hand, we were unable to determinate the provenance of some other commercial resins with close aspect to those of Bursera spp., but which botanical origin seems not to be even in the same Genus, as they are neither triterpenic nor diterpenic resins. It is possible then that these commercial resins are either pure or a mixture of resinous materials, coming from Rhus spp., Liquidambar styraciflua, Myroxylon balsamum and/or Bursera. spp.

Perspectives

The results on the molecular characterization of resins from certified botanical origin open the possibility of applying these results to the analysis of archeological organic materials found in censers, or other type of recipients, to the adhesives used in mosaics or in dental incrustations, to figurines molded in resins and generally speaking to organic materials assimilate to copal. We hope that these results will be applicable not only to Aztec archeological sites and objects, but to the numerous sites from cultures that were located in the middle and the south of Mexican current territory like the Teotihuacans, the Xochicalcans or the Toltecs.

Additionally the determination of the botanical origin of commercial and archaeological samples, using protocols involving PCA and LDA coupled to classical analytical techniques open the possibility of applying similar protocols to two types of resins:

A) The first one constituted by certified resins from Mexican origin. As we said in the second chapter of this work, the great diversity of Busera spp. in Pacific coast lead us to select a number of species for our study, so as a continuation of the present work, some other species may be studied. A special emphasis could be put selecting samples from trees of Busera spp. present in Yucatan peninsula, in order to find the botanical origin of Maya sample.

B) The second possibility is applying this same methodology to samples from other botanical origins like mastic, dammar, oliban, etc. The advantages of using FTIR into patrimony objects are undeniable, as this technique is considered as non destructive, it is fast and generally speaking inexpensive compared to other techniques. This tool could be very useful specifically in the assessment of botanical origin for commercial samples used in art works or in conservation treatments. The possibility of evaluate the preservation state of a commercial sample is a plus.

Concerning molecular profiling of copal composition a very interesting job on the isolation and the structural characterization of compounds present in both fresh and aged resin is to be done.

In the case of molecules that seem to be present only in one species, molecular characterization is really important as they could be use as molecular markers.

Structural characterization of the oxidized products of original compounds may lead important information on degradation pathways that will ease conservator's job.

Finally an inventory of molecules that compose volatile fraction of Mexican copal could be very valuable for perfume industrial uses, as these species seem to have a very rich tenure on sesquiand monoterpenic compounds.

Coupling the information on volatile phase to the information on triterpenic fraction may allow building an extra efficient model for the classification of commercial resins.

Finally another intriguing question that remains open relies on the origin of commercial resins

with such a different composition that no apparent relation to Bursera species could be established.

Materials and

Methods

Materials and Methods

FTIR methodology

The analyzed samples being natural, hard and elastic resins were analyzed, in solid phase. They were prepared as a potassium bromide pellet. The solid samples were crushed in a homogeneous way in an agate mortar with anhydrous potassium bromide (in a proportion of 1/20) transparent to the infrared radiations in the zone ranging between 4.000 and 400 cm -1 . The powder was then compressed under a pressure of 10 t /cm 2 with a manual press, to form thin pellets of translucent aspect.

The pellet was then analyzed, the air being taken as reference. The ratio of the sample to potassium bromide is important; little sample is needed (around 2 or 3 mg) of sample with 200 mg of KBr. Contributions due to moist in the alkali halide (Uvasol, Merck) were avoided, by keeping it desiccated and warm prior to use.

Analytical conditions for HPLC study

For the HPLC study, both standards and global resins were solved in methanol (analytical grade, Merck), the protocol consist in solving a variable amount of crude resin (depending on its botanical origin (refer to table 11) in 2 mL of this solvent, then the solution was sonicated during 10 min. After centrifugation, the supernatant was injected into HPLC system. Each sample was injected in triplicate.

The identification of commercial and archaeological resins was achieved by using a reverse phase column with an elution gradient. The type of column used in this type of research is essential, taking as antecedent the results obtained by our team. Indeed, previous works (Mathe, 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 is carried out in a Waters liquid chromatography consisting of a high-pressure ternary pump Waters 600, a vacuum degasser, a high pressure manual injector valve (20 µL injection loop) and a photodiode array detection (PDA) system Waters 2996. The system is controlled by Empower 2 software. The LC separation is performed at 35° C with a mobile phase consisiting of a binary elution composed of methanol analytical grade (Merck) (A) and bidistilled water (B) containing 0.01% trifluoroacetic acid (TFA), pH = 3 (Acros Organics).

Chromatography is carried out for 35 min at a continuous flow-rate of 1 mL/min. The gradient program is presented in table 24.The chromatograms are acquired at 210 nm. Identification of original triterpenoids was based on the co-injection of samples with reference molecules. NB: This gradient was also used to analyze some samples once an automatic injector was adapted to the HPLC instrumentation.

General methodology for the selective extraction of triterpens by SPE

For SPE Chromabond C-18EC cartridges were chosen. Selection was based upon adsorbents affinity to triterpen molecules. The volume of the cartouche was defined by the volume of the sample and the quantity of analyte present in the crude resin. In the present case for the analysis of commercial resins a 6 mL cartridges was selected, while a 1 mL was chosen for archaeological samples.

Methanol used in this protocol was analytic grade (Merck).

For the 6 mL cartridges SPE protocol steps were as follows 5) Conditioning. For this stage column was conditioned prior to use by drawing 2 times 3 mL of methanol, followed by the same volume of distillated water.

6) Deposit of the sample. A prepared volume of 2 mL of a solution with a concentration of 2 mg/mL of resin solved in methanol was loaded onto and drawn through the column 7) Washing of the adsorbent was performed with 3 mL of a mixture methanol-water (5:95, v/v). The cover of the manifold was then removed and the cover was wiped with a tissue, to remove drops of washing solution.

8) Elution. Triterpenoids were then eluted from the column with 2 x 2 mL of methanol.

For 1 mL cartridges SPE protocol steps were as follows 1) Conditioning. For this stage column was conditioned prior to use by drawing 2 times 300 µL of methanol, followed by the same volume of distillated water.

2) Deposit of the sample. A prepared volume of 200 µL of a solution with a concentration of 2 mg/mL of resin solved in methanol was loaded onto and drawn through the column 3) Washing of the adsorbent was performed with 300 µL of a mixture methanol-water (5:95, v/v). The cover of the manifold was then removed and the cover was wiped with a tissue, to remove drops of washing solution.

165 4) Elution. Triterpenoids were then eluted from the column with 2 x 200 µL of methanol.

After this procedure the eluate collected was analysed by means of HPLC.

GC-MS Materials and Methods

GC

Preparation of samples: off-line derivatization

The derivatization procedure was based on a previous methodology for the analysis of diterpenoid resins developed in our laboratory (Mathe, 2003): a sample of raw resin was crushed, then solved in 0.5mL of pyridine, then it was trimethylsilylated with 0.45 mL of HMDS and 0.3mL of TMSCl. The reaction was conducted at room temperature for 30 min and then the solution was dried with a stream of nitrogen. The residue was immediately dissolved in 2 mL of diethyl ether and filtered on a membrane filter. Then the sample was injected into GC-MS system. 3438.61 2946.77 1708.96 1641.57 1455.57 1380.24 1243.33 1067.63 881 663.75 546.79 ATZM1 3395.53 2945.8 1713.46 1641.59 1455.4 1379.85 1243.22 1067.67 881.1 663.98 547.32 CHOB1 3399.41 2945.39 1709.89 1642.73 1457.92 1379.86 1243.62 1067.64 881.2 663.8 546.91 ATZB1 3400.23 2945.68 1709.71 1642.22 1455.33 1380.43 1242.76 1067.55 880.7 664.12 547.2 0.000000 A3 0.000000 0.000000 A2 0.000000 0.000000 0.000000 A1 0.000000 0.000000 0.000000 0.005339 B11 0.000000 0.000000 0.000000 0.000226 0.001115 B12 0.000000 0.000000 0.000000 0.000000 0.000000 P1 0.000000 0.000000 0.000000 -0.000833 0.000068 P2 0.000000 0.000000 0.000000 -0.000928 -0.000310 B14 0.000000 0.000000 0.000000 -0.000091 0.000676 B15 0.000000 0.000000 0.000000 0.000000 0.000000 E1 0.000000 0.000000 0.000000 0.000000 0.000000 D1 0.000000 0.000000 0.000000 0.000000 0.000000 T3 0.000000 0.000000 0.000000 0.000000 0.000000 Epi lupeol 0.000000 0.000000 0.000000 -0.000258 -0.000183 lupeol 0.000000 0.000000 0.000000 -0.000445 -0.000396 Epi βamyrina 0.000000 0.000000 0.000000 -0.000237 -0.000372 lupenone 0.000000 0.000000 0.000000 -0.000474 -0.000141 Epi αamyrin 0.000000 0.000000 0.000000 -0.000168 -0.000108 β amyrin 0.000000 0.000000 0.000000 -0.000625 -0.000422 αamyrin/β amyrona 0.000000 0.000000 0.000000 -0.001374 -0.000049 α amyrone 0.000000 0.000000 0.000000 -0.000133 -0.000104 T1 0.000000 0.000000 0.000000 0.000000 0.000000 P2 0.000411 B14 -0.000078 0.000500 B15 0.000000 0.000000 0.000000 E1 0.000000 0.000000 0.000000 0.000000 D1 0.000000 0.000000 0.000000 0.000000 0.000000 T3 0.000000 0.000000 0.000000 0.000000 0.000000 Epi lupeol 0.000025 -0.000125 0.000000 0.000000 0.000000 lupeol 0.000014 -0.000295 0.000000 0.000000 0.000000

Bibliographical References

  contributed to this research with samples from the paleo-botanical collection of the INAH. I acknowledge National Mexican Council of Science and Technology (CONACYT) for the Ph D. scholarship that granted me. I acknowledge the Academic body of biomaterials from the Autonomous University of Sinaloa., for their participation in the collection of certified resins.

16 Fig 9 FigFigFigFigFig 39 FigFigFig 52 FigFigFigFigFigFigFig 74 Fig 74 Fig

 16939527474 Fig 1 Pages from the "De la Cruz-Badiano" codex 5 Fig 2 Botanical classification of resin producing trees 7 Fig 3 Phylogeny of Mexican Bursera based on ribosomal A.D.N. 8 Fig 4 Map of Mexico, with the number of Bursera species by region 9 Fig 5 Map with most important Mayan cities 10 Fig 6 Map of Chichén Itzá City 11 Fig 7 The seven stages of "Templo Mayor" a) Aveni, 1988 b) Picture from the National Museum of Anthropology and History, Mexico City 14

Fig 3 Fig 4

 34 Fig 3 Phylogeny of Mexican Bursera based on ribosomal D.N.A. (Becerra and Venable, 1999)

Fig 6

 6 Fig 6 Map of Chichén Itzá City(Cobos, 1997)

  to Mexico Valley the Tecpanecs from Azcapotzalco dominated the region, after a short period they established in Chapultepec where they were expelled. Then they settled in Tizapan a territory dominated by Culhuacan, they abandoned this settlement because of the hostile environment. Finally they took the risk of resettle in a territory under the Tecpanecs control. A little island at Occident of Texcoco lake, in the year 1325 A.D. (year 2 Calli on Aztec calendar). This settlement implicated the subordination to Atzcapotzalco and the payment of tributes. This situation continued until the year 1428 A.D., when Aztec organized, along with Texcoco and Tlacolpan the "triple alliance", which finally defeated Azcapotzalco.

Fig 7

 7 Fig 7 The seven stages of "Templo Mayor" a) Aveni, 1988 b) Picture from the National Museum of Anthropology and History, Mexico City

Fig 8 .

 8 Fig 8. Primitive representation of Quetzalcoatl holding a copal bag on his hand

Fig 9 Archaeological

 9 Fig 9 Archaeological Aztec pieces where copal is believed to be used as adhesive

  Mexican "copal". It is worthy to mention here, the works from De la Cruz-Cañizares in 2005 and Hernandez-Vazquez in 2010, which analyzed commercial samples of resin from Mexico. As well as the work of Stacey and Cartwright 2006, who performed studies in objects from the British Museum collection and the study of a mosaic disc from Magar and Menar in 1995.

  figure 10, for a detailed account on MVA pathway refer to[START_REF] Eisenreich | Deoxycelulose phosphate pathway to terpenoids[END_REF] on MEP to Kashara et al, 2002

  Fig 11 Structure of typical occurring monoterpens (from I to XV) and sesquiterpens (XVI to XXIV) in resins from Burseraceae family. I verbene, II o-cimene, III α-pinene , IV camphene, V β-pinene, VI α-phellandrene, VII α-terpinene, VIII limonene , IX γ-terpinene X α-terpinolene , XI verbenone, XII carvacrol, XIII sabinol, XIV 4-terpineol, XV carvacrol methyl ether, XVI isoledene, XX fenchyl acetate, XXI cis-calamenene, XXII trans-caryophyllene, XXIII caryophyllene oxide, XVIV elemicin: a compound appearing in the monoterpenoid and sesquiterpenoid intervals (De la Cruz Canizares, 2005).

Fig 12 Fig 13

 1213 Fig 12 Triterpenic skeleton of ursane type

Fig

  Fig 14 α-amyrin, α-amyrone and 3-epi-α-amyrin

Fig

  Fig 15 β-amyrin, β-amyrone and 3-epi-β-amyrin

Fig 16

 16 Fig 16 Lupan compounds founded in commercial or archeological resins, described by several authors

Fig 17

 17 Fig 17 Structure of lup-20 (29)-en 3β-23-diol from B. simaruba resin ( Peraza Sanchez, 1995)

Fig 18

 18 Fig 18 Pictures of commercial copals with different colors from unknown botanical origin, taken at our laboratory

Fig 20

 20 Fig 20 Physical aspect of copals from certified botanical origin.

Fig 21

 21 Fig 21 Picture of tree Aztec figurines made of copal, the first taken from Lehman, 1948 and the second and third from Lona-Naoli, 2004

Fig 22

 22 Fig 22 Picture of an Aztec offering 120. (Alonso A. et al, 2008)

  the "complementary opposition" of cosmic energies, a ruling idea of the pre-Columbian religion.[START_REF] Jimenez-Badillo | Una aplicación de la teoria de gráficas en arqueología (primera parte)[END_REF].

Fig 23

 23 Fig 23 Offering "U" from Templo Mayor. It is notable the symmetrical disposition of the four copal balls in the corners and a ceremonial knife in the center (drawing from Carrisoza F. at Jimenez-Badillo, 2009)

Fig 24

 24 Fig 24 Reconstruction of the sacred precint of Tenochtitlán after Antonio Serrato Combé. a) Templo Mayor, b) Cuauhxicalco, c) Tzompantli, d) House of the Eagle Warriors order, e) Area explored by the Archaeological project of Templo Mayor.

  Fig 25 From left to right: Piedra del Sol (1790), Coyolxauhqui (1978) and Tlaltecuhtli (2006)

Fig 26

 26 Fig 26 Map of the cenotes at Yucatán peninsula (Pedroza, 2010)

  Fig 27 The cenote of Chichén Itza[START_REF] Gallareta | Los cenotes de la península de Yucatán[END_REF]

Fig 28

 28 Fig 28 Archaeological objects recovered from the sacred cenote of Chichén Itzá: a) golden bell, b) copper ring, c) ceramic vessel, d) gold monk pendant e) fragment of jadeite sculpture f) gold helmet, g) jade carved with glyphs animal shaped figurine, h) animal shaped figure, i) tripod bowl with offering with jade beads j) tripod bowl with offering k) copal ball with small balls of rubber, l) human face m) corn shaped copal (Catalog of Peabody museum, for scales consult catalog online).

Fig 29

 29 Fig 29 Map of Mexico. Orange zones represent the regions from wich commercial samples were collected and studied, green region plus orange region correspond to geographical extention of Aztec empire.

  Fig 30 A description of the general situation encountered when analyzing historical materials and, a general approach to their study.

  the advantage of being more easily interpretable by non chemist professionals, like conservators and archaeologist. Statistical results also allowed to compare the performance of the classification correlated to the analytic technique used (FTIR and HPLC).

  Fig 31 Picture of non homogeneous archaeological samples referred A) sample 26, B) sample 173, C) sample TMT, D) sample 84

  Fig 33 TMT sample A) from the surface and B) from the interior

Fig 34

 34 Fig 34 Principal components analysis (PCA) projects data along the directions where the data varies the most[START_REF] Terradez | Principal component analysis[END_REF]

  distribution within the plant. The amount and composition of classes of compounds such as alkaloids, flavonoids, essential oils and, many others are governed by the age of the plant or its parts, by the plant's locality (a geographical component) and its habitat (an ecological component). A solid knowledge of chemical variation is essential. Geographical and ecological variation has two main aspects. It may be the result of the plasticity of individual genotypes (modifications), or of a genetic heterogeneity of plant taxa. Genetical variation in local populations is called genetical polymorphism. Polymorphism is one of the starting points of differentiation: individual variants (inclusive of chemical ones) are able to become new races by migration and selection. Species which comprise several races or subspecies (units with their own combination of characters and area and/or habitats)

  ATR cells allow both liquid and solid samples to be examined.

Fig 35 FTIR

 35 Fig 35 FTIR spectra of different species A) B. bipinnata, B) B. excelsa, C) B. grandifolia, D) B. laxiflora.(Intra species variation in spectra can be remarked).A full page view of each of a comparative of the spectra for each studied species can be seen on annex 2 of this work.

Fig 37

 37 Fig 37 Band interpretation for the studied species : A) B. bipinnata, B) B. excelsa, C) B. grandifolia, D) B. laxiflora, E) B. penicillata, F) B. stenophylla

  Fig 39 Loading plot, variables with a high impact on first component are shown in orange, and variables with a high impact on second component are shown on black.

  Fig 40 Distribution in the hyperspace of the resins samples from botanical certified origin, using the first two components with two less variables in the matrix (band positions f and e). Legend different species are shown as follows: •B. laxiflora, B. excelsa, B. stenophylla, B. bipinnata, B. grandifloia, B. penicillata.

Table 9

 9 Results for LDA classification model: Fitting and validation matrix were identical. Rows represent the true class, and columns the assigned class. Wrong assignments are shown in red.

  Fig 41 Distribution in the hyperspace of the resins samples from botanical certified origin, using the first two components, plus a certified sample from • B. copallifera. Legend different species are shown as follows: •B. laxiflora, B. excelsa, B. stenophylla,

  Fig 42 Distribution in the hyperspace of the resins samples from botanical certified origin, using the first two components plus commercial sample • MEXJ1. Legend different species are shown as follows: •B. laxiflora, B. excelsa, B. stenophylla, B. bipinnata, B. grandifloia, B. penicillata.

  Fig 47 Distribution in the hyperspace of the resins samples from botanical certified origin, using the first two components plus FTIR data from archaeological samples: mathematical model was able to discriminate archaeological samples, from different archaeological sites. • Sample from Chcihén Itzá, • Samples from Templo Mayor. Legend different species are shown as follows: •B. laxiflora, B. excelsa, B. stenophylla, B. bipinnata, B. grandifloia, B. penicillata.

Fig 50

 50 Fig 49 Comparative of FTIR spectra offresh andlight aged sample of Huitzuco (commercial sample).

Fig 52

 52 Fig 51 Distribution in the hyperspace of the resins samples from botanical certified origin, using the first two components plus samples of HUITZUCO fresh and aged. Legend different species are shown as follows: •B. laxiflora, B. excelsa, B. stenophylla, B. bipinnata, B. grandifloia, B. penicillata, fresh Huitzuco sample • aged Huitzuco sample.

Fig 53

 53 Fig 53 Comparative of FTIR spectra of theinner part of TMT andthe more degradated surface of TMT

  Fig 54 Distribution in the hyperspace of the resins samples from botanical certified origin, using the first two components plus TMT samples from its inner part and its surface. Legend different species are shown as follows: •B. laxiflora, B. excelsa, B. stenophylla, B. bipinnata, B. grandifloia, B. penicillata • TMT sample from the inner part, •TMT sample from the surface.

  threes, the archaeological Aztec resins may have their origin between B. bipinnta and ANALYTICAL STUDY OF THE RESINS AND DISCUSSION ANALYTICAL STUDY OF THE RESINS AND DISCUSSION 87 B.stenophylla. This turns FTIR spectroscopy in an invaluable tool for the conservator professionals implicated in archaeological works.Assessing of the botanical origin of resins used in restoration of archaeological objects or even paintings is a must.As stated by Dietemann in his work from 2003, "With increasing compositional complexity the reactivity of the reference substances increases, thus even greater reactivity is then expected in natural resins." Therefore mixture of resins from different botanical origins may modify the original materials of a patrimony object and even worst it can result in accelerated degradation of them. In the authentication field, this method can be also used, as it has discriminated botanical origin for archaeological samples from different archaeological sites. been applied in several laboratories specialized in artworks.

Fig 56

 56 Fig 56 Chromatogram of B. bipinnata (sample 11d2), triterpenic zone running from 24 to 35 min in retention time

Fig 57

 57 Fig 57 Chromatograms for certified resins. Identification of triterpenic compounds is as follows: 1) 3-epi-lupeol,

  Figure 57 presents the chromatogram for each species and an account on the detection of the major peaks for both polar and triterpenic molecules. When looking to these chromatograms, we conclude that the closest tritrepenic molecular composition in qualitative terms is found between B. bipinnata and B. stenophylla. A difference between B. bipinnata and B. stenophylla, is the absence of the compound T3 at 25.4 min in B.

Fig 59

 59 Fig 59 Chromatograms of samples type A and B of B. stenophylla. Identification of triterpenic compounds is as follows: 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

Fig 60

 60 Fig 60 Molecular profile types 1 and 2 of B. excelsa. . Identification of triterpenic compounds is as follows: 1) 3epi-lupeol, 2) lupeol, 4) lupenone.

Fig 61 B

 61 Fig 61 B. simaruba lot 1 with a richer polar fraction than lot 2. Identification of triterpenic compounds is as follows: 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

Fig 63

 63 Fig 63 Chromatograms of ATZB1 and Huitzuco commercial samples that have the same molecular composition than B. bipinnata samples.. Identification of triterpenic compounds is as follows: 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

Fig 64

 64 figure64). As they present a molecular profile similar to that of this species.

Fig 67

 67 Fig 67 Chromatograms of archeological sample with molecular profile A.Identification of triterpenic compounds is as follows: 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

  Fig 68 Chromatograms from archeological samples with B profile from Templo Mayor. Identification of triterpenic compounds is as follows: 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

Fig 69

 69 Fig 69 Chromatogram of Chichén Itzá archeological sample.Identification of triterpenic compounds is as follows: 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

Fig 70

 70 Fig 70 Distribution in the hyperspace of the resins samples from botanical certified origin, using the first two components, with HPLC data. Species are shown as follows: •B. laxiflora, B. excelsa, B. stenophylla, B.

Fig 71

 71 Fig 71 Loading plot for PCA analysis using HPLC data for botanical certified resins

Fig

  Fig 75 Schematic view of GC instrumentation and their components

  2004. Using silylation reagents like hexamethyldisilazane (HMDS) and chlorotrimethylsilane (TMSCl) presents the advantage of derivatizing in a single and fast step both carboxylic and hydroxyl groups. This enabled a more complete characterization of the composition of resins. Indeed, the presence of trimethylsilylated (TMS) compounds induced the formation of characteristics ions. For molecules containing a hydroxyl group, it is important to note the existence of fragments m/z = 73 and m/z = 75 (Fig 76).

Fig 76

 76 Fig 76 Characteristic fragments from silylated compoundsIf the molecule counts on its structure more than one hydroxyl group either from an alcohol or a carboxylic acid functions, the presence of an ion m/z = 147 resulting from the addition of the two previous fragments is observed.

  , β-OTMS ether 3β-hydroxy-urs-12-en-3-ol, β-OTMS ether U α-H, β-OH 3-epi-α-amyrin, α-OTMS ether 3α-hydroxy-urs-12-en-3-ol, α-OTMS ether U α-OH, β-H β-amyrenone olean-12-en-3-one O O β-amyrin, β-OTMS ether 3β-hydroxy-olean-12-en-3-ol, β-OTMS ether O α-H, β-OH 3-epi-β-amyrin, α-OTMS ether 3α-hydroxy-olean-12-en-3-ol, α-OTMS ether O OH, β-H lupeol, β-OTMS ether 3β-lup-20(29)-en-3-ol, β-OTMS ether L α-H, β-OH 3-epi-lupeol, α-OTMS ether 3α-lup-20(29)-en-3-ol, α-OTMS ether L αcompound was attributed a roman number. This notation is used along the present chapter, in the chromatograms, to identify these molecules.

  , the retention time of the compounds was influenced by the number and the type of functional groups present, and generally increased with increasing molecular weight. Concerning the chemical skeleton in the retention mechanism, lupane standards are retained longer than their ursane isomers and even longer than oleanane ones. In the other hand the absolute configuration of C-3 has a great influence into retention times: β-configuration gave a longer retention time than α-configuration. This fact allows to differentiate β -amyrin (V), α-amyrin (VII) and lupeol (IX), from their respective 3-epimer (I, II and III) in the chromatogram. Under the chromatographic conditions ANALYTICAL STUDY OF THE RESINS AND DISCUSSION ANALYTICAL STUDY OF THE RESINS AND DISCUSSION PAOLA LUCERO 128

Fig

  Fig 81 GC chromatogram of B. bipinnata sample 11c1

  in B. excelsa composition are identified with a letter "E" plus a progressive number. The determination of the exact structure of these compounds is beyond the scope of this research. Its study, depending on their specificity these molecules could be used as molecular markers for resins with this botanical origin.

Fig 83

 83 Fig 83 Zoom into triterpenic zone of GC chromatogram of a sample of B. excelsa (27a4). Identification of triterpenic compounds is as follows: (I) 3-epi-β-amyrin α-OTMS, (II) 3-epi-α-amyrin α-OTMS, (III) 3-epi-lupeol α-OTMS.

Fig 84

 84 Fig 84 Zoom into triterpenic zone of GC chromatogram of sample of B. grandifolia (34b1Q). Identification of triterpenic compounds is as follows: (I) 3-epi-β-amyrin α-OTMS, (II) 3-epi-α-amyrin α-OTMS, (IV) β-amyrone (VI) α-amyrone.

Fig 85

 85 Fig 85 Zoom into triterpenic zone of GC chromatogram of sample of a B. laxiflora (47a1Q). Identification of triterpenic compounds is as follows: (I) 3-epi-β-amyrin α-OTMS, (II) 3-epi-α-amyrin α-OTMS, (III) 3-epi-lupeol α-OTMS, (IV) β-amyrone, (V) β-amyrin β-OTMS, (VIII) lupeol β-OTMS, (IX) lupenone.

Fig 86

 86 Fig 86 Zoom into triterpenic zone of GC chromatogram of a sample of B. laxiflora (47b1Q). Identification of triterpenic compounds is as follows: (I) 3-epi-β-amyrin α-OTMS, (II) 3-epi-α-amyrin α-OTMS, (III) 3-epi-lupeol α-OTMS, (IV) β-amyrone, (V) β-amyrin β-OTMS, (VIII) lupeol β-OTMS, (IX) lupenone

Fig 87

 87 Fig 87 Zoom into first triterpenic zone of GC chromatogram of a sample of B. penicillata (63a4).Identification of triterpenic compounds is as follows: (I) 3-epi-β-amyrin α-OTMS, (II) 3-epi-α-amyrin α-OTMS, (III) 3-epi-lupeol α-OTMS, (IV) β-amyrone, (V) β-amyrin β-OTMS, (VII) α-amyrin β-OTMS, (VIII) lupeol.

Fig 88

 88 Fig 88 Zoom into second triterpenic zone of GC chromatogram of sample of B. penicillata (63a4).

Fig 89

 89 Fig 89 Zoom into triterpenic zone of GC chromatogram of a sample of B. simaruba (70a1Q). Identification of triterpenic compounds is as follows: (I) 3-epi-β-amyrin α-OTMS, (II) 3-epi-α-amyrin α-OTMS, (III) 3-epi-lupeol α-OTMS, (IV) β-amyrone, (V) β-amyrin β-OTMS, (VI) α-amyrone, (VII) α-amyrin β-OTMS, (VIII) lupeol.

Fig 90

 90 Fig 90 Zoom into triterpenic zone of GC chromatogram of the B. copallifera resin sample. Identification of triterpenic compounds is as follows: (I) 3-epi-β-amyrin α-OTMS, (II) 3-epi-α-amyrin α-OTMS, (III) 3-epi-lupeol α-OTMS, (IV) β-amyrone, (V) β-amyrin β-OTMS, (VI) α-amyrone, (VII) α-amyrin β-OTMS, (VIII) lupenone, (IX) lupeol β-OTMS

  Fig 91 A) complete GC chromatogram and B) Zoom into triterpenic zone of GC chromatogram of archaeological sample 26. Identification of triterpenic compounds is as follows: (I) 3-epi-β-amyrin α-OTMS, (II) 3-epi-α-amyrin α-OTMS, (III) 3-epi-lupeol α-OTMS, (IV) β-amyrone, (V) β-amyrin β-OTMS, (VI) α-amyrone, (VII) α-amyrin β-OTMS, (VIII) lupeol β-OTMS, (IX) lupenone

Fig 93 A )

 A Fig 93 A) Complete chromatogram and B) zoom into triterpenic zone of archaeological sample 52. Identification of triterpenic compounds is as follows: (I) 3-epi-β-amyrin α-OTMS, (II) 3-epi-α-amyrin α-OTMS, (III) 3-epi-lupeol α-OTMS, (IV) β-amyrone, (V) β-amyrin β-OTMS, (VI) α-amyrone, (VII) α-amyrin β-OTMS, (VIII) lupeol β-OTMS, (IX) lupenone.

Fig 94 A )

 A Fig 94 A) Complete chromatogram and B) zoom into triterpenic zone of archaeological sample 84. Identification of triterpenic compounds is as follows: (I) 3-epi-β-amyrin α-OTMS, (II) 3-epi-α-amyrin α-OTMS, (III) 3-epi-lupeol α-OTMS, (IV) β-amyrone, (V) β-amyrin β-OTMS, (VI) α-amyrone, (VII) α-amyrin β-OTMS, (VIII) lupeol β-OTMS, (IX) lupenone.

  Fig 96 A) Complete chromatogram and B) zoom into triterpenic zone of archaeological sample TMT. Identification of triterpenic compounds is as follows: (I) 3-epi-β-amyrin α-OTMS, (II) 3-epi-α-amyrin α-OTMS, (III) 3-epi-lupeol α-OTMS, (IV) β-amyrone, (V) β-amyrin β-OTMS, (VI) α-amyrone, (VII) α-amyrin β-OTMS, (VIII) lupeol β-OTMS, (IX) lupenone.

  Fig 97 A) Complete chromatogram and B) zoom into triterpenic zone of archaeological sample M1. Identification of triterpenic compounds is as follows: (I) 3-epi-β-amyrin α-OTMS, (II) 3-epi-α-amyrin α-OTMS, (III) 3-epi-lupeol α-OTMS, (IV) β-amyrone, (V) β-amyrin β-OTMS, (VI) α-amyrone, (VII) α-amyrin β-OTMS, (VIII) lupeol β-OTMS, (IX) lupenone.

  inferior compared to their proportion in the Aztec archaeological samples.

  III) 3-epi-lupeol α-OTMS, (IV) β-amyrone, (V) β-amyrin β-OTMS, (VI) α-amyrone, (VII) α-amyrin β-OTMS, (VIII) lupeol β-OTMS.

  Fig 99 A) Complete chromatogram and B) zoom into triterpenic zone of commercial sample Sidral. Identification of triterpenic compounds is as follows: (I) 3-epi-β-amyrin α-OTMS, (II) 3-epi-α-amyrin α-OTMS, (III) 3-epi-lupeol α-OTMS, (IV) β-amyrone, (V) β-amyrin β-OTMS, (VI) α-amyrone, (VII) α-amyrin β-OTMS, (VIII) lupenone, (IX) lupeol β-OTMS

Fig 100

 100 Fig 100 Zoom into triterpenic zone of commercial sample SONB4. Identification of triterpenic compounds is as follows: (I) 3-epi-β-amyrin α-OTMS, (II) 3-epi-α-amyrin α-OTMS, (III) 3-epi-lupeol α-OTMS, (IV) β-amyrone, (V) β-amyrin β-OTMS, (VI) α-amyrone, (VII) α-amyrin β-OTMS, (VIII) lupeol β-OTMS, (IX) lupenone.

  Fig 101 Global Chromatograms for SONB4 and ATZJ2

Fig

  Fig 102 Global Chromatogram for Oaxc2

  Fig 103 Zoom into triterpenic zones of the chromatograms for A) IZUP1 and B) SONR1 Identification of triterpenic compounds is as follows: (I) 3-epi-β-amyrin α-OTMS, (II) 3-epi-α-amyrin α-OTMS, (III) 3-epi-lupeol α-OTMS, (IV) β-amyrone, (V) β-amyrin β-OTMS, (VI) α-amyrone, (VII) α-amyrin β-OTMS, (VIII) lupeol β-OTMS, (IX) lupenone.

  Fig 104 Global chromatograms for A) IZUP1 and B) SONR1

Fig 106

 106 Fig 105 Global chromatograms for A) SONB4 resin and B) SONB4 aged resin

Fig 107

 107 Fig 107 Zoom into triterpenic zone of chromatograms of TMT resin in the inner part and from the surface. Identification of triterpenic compounds is as follows: (I) 3-epi-β-amyrin α-OTMS, (II) 3-epi-α-amyrin α-OTMS, (III) 3-epi-lupeol α-OTMS, (IV) β-amyrone, (V) β-amyrin β-OTMS, (VI) α-amyrone, (VII) α-amyrin β-OTMS, (VIII) lupeol β-OTMS, (IX) lupenone.

Fig 108

 108 Fig 108 Mass fragmentation for the "N" compound, peak detected at 39.78 min from resin TMT surface.

  BS1 and BS2 are actually molecules formed fast upon oxidation. This would explain the augmentation on their proportions in more aged resins.

  -MS was carried out in a Varian Saturn 3900 gas chromatography, with a Varian injector 1177 coupled with a Varian 2100 T ion trap mass spectrometer (Varian Walnut Creek, USA). The gas chromatograph was equipped with a 30 m length, and 0,25 mm internal diameter fused silica capillary column coated with 0.25 µm film of poly (5% phenyl, 95% dimethylsiloxane): CP-Sil 8 CB Low Bleed/MS (Varian). The MS electron multiplier voltage was set at 1400 V and an ionization time of 25000 µs was used, running in electronic impact (EI) with transfer line, ion trap and manifold temperatures of 300°C, 200°C and 50°C respectively. The mass spectrometer was set to scan masses ranging between 40 and 650 m/z with an ionizing voltage of 70 eV.Samples were injected (1 µL) with no splitting ratio. The injector temperature was set to 250°C.A continuous flow rate of 1 mL/min of chromatographic grade helium was used. Temperature gradient employed for the global study of the resins is shown in table 26.

Fig

  Fig 109 Map of the GPS position of originating trees for certified resins

Fig

  Fig 110 FTIR spectra of B. bipinnata samples

Fig

  Fig 122 Mass spectra of molecular markers for B. copallifera

Table 10

 10 Amount of resins used for the HPLC chromatographic study 90 Table11Gum and resin percentage in resin samples from studied species 91 Table12Unsoluble matter in apolar solvents in archeological samples.

92 Table 13 Arqueological Aztec samples 93 Table 14

  Archeological sample from Chichén Itzá 94 Table 15 Unsoluble matter in apolar solvents in archeological samples 95 Table

Table 19

 19 Validation matrix of LDA, Rows represent the true class and the columns the assigned class

	XI

Table

  TMS structure of the triterpenic standard molecules studied by GC-MS.

O: Oleanane, U:Ursane, L: Lupane 121

Table

  Time of retention of standard molecules under the designed GC-MS gradients

			122
	Table Amount of resins used for the GC-MS study	123
	Conclusions and Perspectives
	Table Recapitulative of botanical origin for archaeological and commercial	155
		samples studied
	Materials and Methods
	Table	HPLC Gradient 1, employed for the study of the terpenic compounds in fresh resins	162
	Table	HPLC gradient 2 employed for the study of the terpenic compounds in archaeological resins	162
	Table	Gradient 1, GC-MS temperature gradient for the global analysis of resins	164

Table Gradient

 Gradient

		LISTE OF TABLES
			188
	Table	Pictures of some commercial samples	190
	Table	Selected pictures of optical microscopic study of certified origin	191
		samples from B. bipinnata species
	Table	Selected pictures of optical microscopic study of certified origin	192
		samples from B. bipinnata species, and from archeological sample
		M1
	Table	Selected pictures of electronic microscopic study of certified origin	193
		samples from B. Bipinnata species
	Table	Selected pictures of electronic microscopic study of certified origin	194
		samples from B. bipinnata species
	Table	Selected pictures of electronic microscopic study of certified origin	195
		samples from B. bipinnata species
	Table	Picture of Sidral sample before and after light ageing	196
	Table	Botanical differences between B. bipinnata and B. stenophylla	197
	Table	FTIR bands of certified resins used in PCA	206
	Table	FTIR bands of archaeological resins used in PCA	207
	Table	LDA results of classification of FTIR data of certified resins	208
	Table	FTIR data used on PCA on selected commercial sample	208
			XII

2, optimal gradient for the analysis of triterpenoids GC-MS 165

ANNEXES

Table

Table of certified copals B. bipinnata, B. excelsa and B. grandifolia, with GPS position of originating tree and date of collection 186 Table Table of B. laxiflora y B. penicillata certified copals, with GPS position of originating tree and date of collection 187 Table Table of B. simaruba, B. stenophylla certified copals, with GPS position of originating tree and date of collection

Table 43

 43 HPLC data of peaks corresponding to non triterpenic compounds in botanical certified resins and archaeological resins used in PCA Table 44 HPLC data of peaks corresponding to non triterpenic compounds in botanical certified resins and archaeological resins used in PCA Table 45 HPLC data of peaks corresponding to non triterpenic compounds in botanical certified resins and archaeological resins used in PCA

Table 46

 46

Table 53

 53 Mass spectra and retention time for identified triterpenes in Mexican copal

PAOLA LUCERO

11

  refer to the natural resins, produced by New World trees, that were burnt in religious ceremonies, probably because of the parallel use in Europe of Boswellia resins.Mesoamerican cultures considered copal as a protector. It was called "iztacteteo" or "White gods"; this material was believed to have magic and religious powers. After the Spanish chronicles from Bernal Díaz del Castillo, BernardinoSahagún and Diego Landa, ancient

	CHAPTER I	GENERAL PRESENTATION OF COPALS
	India) to
	PAOLA LUCERO	19

  or more trees". It is also noteworthy to say that in some regions, like in the Mexican community of Teotlalco in Puebla up to six species ofBursera (B. bipinnata, B.

	CHAPTER I	GENERAL PRESENTATION OF COPALS
	recipient for two
	PAOLA LUCERO	22

jorullensis, B.odorata, B. copallifera, B. bicolor, B. glabrifolia)

can coexist (Garcia-Hernandez,

Table 1 Monosaccharide composition of B. Simaruba (Magaloni, 1996)

 1

	CHAPTER I

Presentation of the copal samples and the analytical strategy CHAPTER II PRESENTATION OF THE ANALYTICAL STRATEGY AND OF THE COPAL SAMPLES PAOLA LUCERO

Scale 1 cm picture: 2cm 1cm picture: 15cm 1cm picture: 20 cm 1cm picture: 1cm Fig 19 Images from left to righ: white copal, "piedra" copal at Cholula market, yellow "lágrima" copal at Izúcar market, and "mirra" at Azcapotzalco market. CHAPTER II PRESENTATION OF THE ANALYTICAL STRATEGY AND OF THE COPAL SAMPLES PAOLA LUCERO 34 2.2 Resins from botanical certified origin 2.2.1 Botanical References

	For our research we included 8 species, among the 80 different Bursera trees in Mexico.
	Here we present the botanical references for each species:
	B. bipinnata (DC.) Engl.(B. elemifera (Royle) Bail, B. gracilis Engl.). Description in
	Rzedowski and Calderón de Rzedowski (1996b) ; Rzedowski et al. (2004); all with
	ilustrations. Engler (1931) also proposed and ilustration.
	B. copallifera (DC.) Bullock. (B. jorullensis H.B.K., B. palmeri var. glabrescens S. Wats.)
	Description in Toledo Manzur (1982), Guizar-Nolazco and Sanchez-Velez (1991); the last
	work has illustrations as well.
	B. excelsa (H.B.K.) Engl. (B. sphaerocarpa Sprague & Riley). Plant description in Toledo
	Manzur (1982) and in Pennington and Sarukhan (1998); illustration in the last work and in
	McVaugh y Rzedowski (1965).
	B. grandifolia (Schltdl.) Engl. (Elaphrium occidentale Rose). Description in Toledo Manzur

OF THE ANALYTICAL STRATEGY AND OF THE COPAL SAMPLES PAOLA LUCERO 37 ID 26 51 52 84 173 TMT M1

	CHAPTER II PRESENTATION Artifact number A133 A 252 A260	A957	Unknown	Unknown	A5
			Spiritual	Amorphous	Amorphous	Adhesive	Amorphous	Amorphous	Molding
	Nature of the	material	offering, part of a probably deposed in liquid state, preserved as	material	material	from base of a the pectoral-ceremonial knife	material	material	material from Aztec figurine	an
			conglomerate
	Weight of	sample (g)	0.25	0.04	0.02	0.39	0.32	1.5	2.3
	Number of	Offering	128	126	126	125	120	botanical Collection of the INAH Unknown, Paleo	140
	Picture
	Scale 5 mm: 800x	5 mm: 80x	5mm: 80x	5mm: 25x	5mm: 25x	5mm: 25x	3 mm: 80x
	Aztec god figurine, sample 26 was part of a conglomerate of bars, probably offered liquid
	and contained in maguey leaves. The other samples come from amorphous artifacts.

Table 2 Archaeological Aztec samples N.B. The number appearing as ID for each archaeological sample, on table 2, will be retained all along the present work to refer and differentiate each Aztec resin sample.

CHAPTER II PRESENTATION OF THE ANALYTICAL STRATEGY AND OF THE COPAL SAMPLES PAOLA LUCERO 42 2.4.3 Offering 126

	It was excavated from May 2008.
	It was founded in a 2 m long and 1.1 m width box, constituted by four walls and a cover, from
	flat smoothed plaques of andesite. The interior walls were made of stone covered with clay and
	lime.

OF THE ANALYTICAL STRATEGY AND OF THE COPAL SAMPLES PAOLA LUCERO

  first professional archaeological research project organized by the National Institute of

	Anthropology and History (INAH) in Chichén Itzá took place between 1960 and 1961 under the CHAPTER II direction of Roman Piña Chán. On this occasion, use was made of propelled-air equipment with a suction hose, but researcher soon change the technique for diving as the propelled air equipment was inadequate for the extraction of fragile items. The sample of copal that was analyzed in this work (cf table 6) is likely to be recovered by this archaeological project. It was part of the paleobotanical collection of the INAH, where it has been stored for around 45 years, under museum conditions. It is constituted of a series of yellow PRESENTATION 43 fragments in tear shape and size, mixed with sable from the bottom of the cenote.

ID Photos Total weight of the sample (g) Provenance, additional information Chichén Itzá Fragment of 0.3cm x 0.15cm 23.47 Paleo botanical Collection of the INAH Sample with small fragments of resin mixted to sable from the cenote Table 3 Archaeological sample from Chichén Itzá 2.5.1 The Chichén Itzá Cenote: macroambiental conditions

	Yucatán peninsula is geologically speaking a flat calcareous platform without folding. The
	karstic constitution of Yucatán is mailnly composed on calcium carbonate (CaCO 3 ) limestone
	rock (Martos-López, 2007), with an average elevation of 20 m over the sea level, and tropical
	weather: intense pluvial precipitations, high temperature and dense vegetation are characteristics
	of this zone

CHAPTER II PRESENTATION OF THE ANALYTICAL STRATEGY AND OF THE COPAL SAMPLES PAOLA LUCERO

	defines any underground space with water, regardless its dimensions or characteristics (Martos-
	44

CHAPTER II PRESENTATION OF THE ANALYTICAL STRATEGY AND OF THE COPAL SAMPLES PAOLA LUCERO

Table 4 Copals collected in 2004-2005 Reference Description Color, physical state Geographical origin, year of collection

 4

	Reference	Description	Color, physical state	Geographical origin, year of
					collection
	OaxC1	Copal "lágrima"	Opaque, yellow , solid	Oaxaca, Sep. 2005
	OaxC2	Black copal	Black-redish, solid	Oaxaca, Sep. 2005
	OaxC3	Yellow copal	Brown, sand looking	Oaxaca, Sep. 2005
			powder, solid
	L1	White copal	Transparent, light	Colima, unknown
			yellow,solid
	L2	White copal	Opaque light yellow	Colima, unknown
			powder, solid
	M6	White copal	Transparent light		Texcoco
			yellow powder, solid
	M6Q	White copal	Light yellow, solid		Texcoco
	ATZB1	White copal	Light yellow resin, totally opaque,	Unknown, 2008
			dusty in the surface
	ATZM1	Wooden fragments	Resin: withish, opaque, solid.	Unknown, 2008
		impregnated with resin
	CHOB1	White copal	Light yellow opaque powder.	Puebla, 2008
	CHOB2	White copal	White opaque resin solid	Puebla, 2008
	CHOB3	White copal	Yellow opaque resin, solid.	Puebla, 2008
	CHOB4	White copal	Light yellow resin with translucid	Puebla, 2008
			and opaque localized zones
	CHOM1	Wooden fragments	Resin: withish, opaque, solid.	Puebla, 2008
		impregnated with resin
	CHOR1	Red Copal	Brownish translucid resin with red	Puebla, 2008

Table 5 Copals collected in 2008

 5

	Reference	Description	Colour, physical state	Geographical origin,
				year of collection
	ATZB2	White copal	Light yellow, solid, soft odour	Unkwown, 2010
	ATZJ2	Copal "lagrima"	Colour varies from light opaque	Unkwown, 2010
			yellow to orange, fragments solid
	SONR2	Red copal	into big fragments of around Brownish resin conglomerated	Unkwown, 2010
			200g, dusty surface
	SONB4	White copal	surface. In barrels of 15 cm, quite White translucid resin, shiny	Unkwown, 2010
			sticky, solid.
	MEXB1	White copal	shapped into bars of 20cm long Bright yellow resin, shiny,	Unkwown, 2010
			for 8cm width.
	MEXJ1	Yellow copal	Tears in different colours ranging	Unkwown, 2010
			from translucent yellow to brown
			passing by orange and bright
			yellow.
	MEZM1	Wooden fragments	Hard, bright yellow resin	Unkwown, 2010
		impregnated with resin

Table 6 Copals collected in 2010 CHAPTER II PRESENTATION OF THE ANALYTICAL STRATEGY AND OF THE COPAL SAMPLES PAOLA LUCERO 51

 6

Table 7 Assignment for the most characteristic IR bands of some terpenoic compounds (Schulz and Baranska, 2007)

 7

	Terpenes	Compounds	ATR-IR	Assignment	Occurrence (example)
			(cm -1 )
	Acyclic	Myrcene	1637	ν(C=C)	Pepper (Piper nigrum L.) (fruit)
	monoterpenes		1595
			989	ω(CH2)

Table 8 Band positions taken into account as variables for the PCA matrix.

 8

of bands between 1200 and 1300 cm-1 region. For A) B. bipinnata and B) B. stenophylla. ANALYTICAL STUDY OF THE RESINS AND DISCUSSION ANALYTICAL STUDY OF THE RESINS AND DISCUSSION

	ANALYTICAL STUDY OF THE RESINS AND DISCUSSION
	ANALYTICAL STUDY OF THE RESINS AND DISCUSSION A) B. bipinnata 0.04
	R 2	0.99428
	0.03 5 bands at : 1206.6, 1242.9, 1250.5, 1260.8, 1270.7	Data: IZUM1CORREGID_B
										Model: Lorentz
		0.02							B	Chi^2 = 1.5322E-6
										R^2 = 0.99174
	Y Axis Title	0.02 0.00 Y Axis Title 0.01								Data: A72A1_B Model: Lorentz Chi^2 = 4.213E-7 R^2 = 0.99428 y0 0 ±0 xc1 1270.70959 w1 15.92102 xc3 1254.23897 ±1.81467 ±1.90559 ±3.42698 w3 7.71379 ±7.902 A3 0.0766 ±0.12367 A2 1.2503 ±0.15608 y0 -0.01555 ±0.00317 xc1 1206.92276 ±0.27931 w1 13.90694 ±2.07545 A1 0.44732 ±0.10503 xc2 1243.08685 ±0.40209 w2 18.52401 ±1.5265
		0.00 -0.01								A1 xc2 w2 A2 xc4 1258.88178 ±3.84566 0.19315 ±0.12644 1250.51474 ±1.28473 11.91148 ±8.86639 0.14849 w4 4.84683 ±14.71948 ±0.20933 xc3 1260.79631 ±1.23029 A4 0.01204 ±0.06484
		1320 1300 -0.02	1300 1280	1280	1260 1260 X Axis Title 1240 X Axis Title 1220 1240	1200 1220	1180	1200	w3 A3 xc5 1269.17948 ±1.49953 14.43326 ±9.14298 0.20544 ±0.23967 xc4 1242.93273 ±0.58563 w4 11.7045 ±1.12227 A4 0.36562 w5 31.88003 ±6.09088 A5 1.15292 ±0.35811 ±0.10309 xc5 1206.64098 ±0.75395 w5 7.43576 ±2.24147 A5 0.0304 ±0.00668
	B) B. stenophylla
	R2	0.99888
	4 bands at: 1211.3, 1221.4, 1244.3, 1269.1 0.07
										Data: MEXJ1_B
										Model: Lorentz
		0.06								B
		0.040							Chi^2 = 5.6225E-6
		0.035 0.05 0.030							Data: R11D1CORR_B R^2 = 0.98098 Model: Lorentz Chi^2 = 1.8888E-7 R^2 y0 0 ±0 = 0.99888 xc1 1207.26171 ±0.28818
	Y Axis Title	0.010 0.015 0.020 0.025 0.02 Y Axis Title 0.03 0.04							y0 xc1 w1 A1 xc2 w2 A2 xc3 w3 A3 xc4 w4 A4	-0.00517 1211.30589 16.21574 0.25461 1221.4362 17.30905 0.44573 1244.33831 27.37031 1.57924 1269.05838 18.53873 0.30693 A3 0.12175 w1 11.15069 ±0.00024 ±0.85139 ±1.83919 ±0.07447 ±0.5772 ±1.97577 ±0.09777 ±0.15209 ±0.80145 ±0.06077 ±0.29242 ±1.24322 ±0.02785 ±0.11401 ±0.91069 A1 0.54065 ±0.03402 xc2 1243.0935 ±0.45121 w2 17.00367 ±1.05099 A2 1.59513 ±0.13411 xc3 1254.0217 ±1.59567 w3 9.64244 ±6.50509
		0.005							xc4 1271.8075 ±0.51061
		0.000 0.01							w4 12.19891 A4 0.38531	±1.75407 ±0.04646
		-0.005
		0.00	1320	1300	1280	1260	1240	1220	1200	1180
						X Axis Title
		-0.01
		1300	1280	1260	1240	1220		1200	1180
	76 Fig 44 Deconvolution 77 X Axis Title

Fig 45 IZUM1 deconvolution of 1300-1200 region: botanical origin B. stenophylla R 2 99.17 % Fig 46 MEXJ1 deconvolution of 1300-1200 region: botanical origin B. bipinnata R

2 98.09 % ANALYTICAL STUDY OF THE RESINS AND DISCUSSION ANALYTICAL STUDY OF THE RESINS AND DISCUSSION 78

  When the procedure was applied to commercial resins, MEXJ1 and MEZM1, different botanical origins were attributed to each resin, based upon the number of signals in the mentioned region (figures 45 and 46): R 2 was 99.17% for IZUM1 and R 2 for MEXJ1 was 98.09 % Archaeological samples were included into the analysis, and the graphic for the two first components was made. Here we present it in figure47. From this graph it is important to note that archaeological sample from Chichén Itzá is located in a very distant region than the rest of the samples. Therefore, and considering firstly our previous study with B. copallifera, secondly the distance between both archaeological sites and oral tradition, different botanical origin can be assumed for this sample.

	3.12	Application to archaeological resins

OF THE RESINS AND DISCUSSION ANALYTICAL STUDY OF THE RESINS AND DISCUSSION 81 carbon

  -carbon double bonds and tertiary carbon atoms. These reactions lead to degradation that manifests as yellowing and cracking[START_REF] De La Rié | Photochemical and thermal degradation of films of dammar resin[END_REF].

had shown that Mexican copal contain many of the typical functional groups that promote auto-oxidation reactions, such as carbonyl groups, ANALYTICAL STUDY

Table 11

 11 resumes the percentage of gum (non soluble material in apolar solvents) found in each studied species.

		% Gum	% Resin
	B. bipinnata	0	100
	B. excelsa	0.5	99.5
	B. simaruba	25	75
	B. penicillata	0	100
	B. stenophylla	0	100
	B. laxiflora	8	92
	B. grandifolia	75	25

Table 11 Gum and resin percentage in resin samples from certified species

Table 12 Insoluble matter in apolar solvents in archeological samples

 12

Table 13 Time of retention of standard molecules under the designed HPLC gradient

	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

Table 14 Statistical data from calibration. Chromatographic conditions: detection at 210 nm for standard compounds

 14

	Triterpenic standard	Linearity range (µg/mL)	Calibration equation	LOD (µg/mL)	LOQ (µg/mL)	Correlation coefficient (R 2 )
	α-amyrin	2.75-0200 Y = 24499375.88*X-100187.19	3.52	11.7	0.9993
	α-amyrone	0.6041-290 Y=11485526.05*X+25383.31	2.5	10.6	0.9995
	β-amyrin	2.2-100	Y=116395*X-37759,24	3.7	12.5	0.9992
	β-amyrone	2.1-100	Y=8693057.33*X+31615.5	2.97	9.9	0.9989
	lupeol	0.5-100	Y=8862571*X+40562.44	2.07	6.9	0.9996
	lupenone	0.6-100	Y=11235316.61*X+26491.86	6.25	20.8	0.9995

Table 16 Retention time of peak corresponding to non triterpenic compounds founded in each species (+ presence, -absence) ID of the peak T3 3-epi- lupeol lupeol 3-epi-ß amyrin lupenone 3-epi-α amyrin ß- amyrin α-amyrin/ ß-amyrone α-amyrone T4

	Ret. time (min)	25.5	26.5	27.6	28.2	28.7	29.4	29.9	31.3	32.1	33.5
	B.bipinnata	-	+	+	+	+	+	+	+	+	-
	B. excelsa	+	+	+	-	+	-	+	-	-	-
	B.laxiflora	-	+	+	+	+	-	+	+	+	-
	B.laxiflora	-	+	+	-	+	-	+	+	+	-
	B. penicillata	-	+	+	+	+	+	+	+	+	-
	B. simaruba	-	+	+	+	+	+	+	+	+	-
	B. stenophylla	+	+	+	+	+	+	+	+	+	+

Table 17 Retention time of peaks corresponding to triterpenic compounds found in each species (+ presence, - absence)

 17

	CHAPTER IV
	CHROMATOGRAPHIC TECHNIQUES: HPLC UV/VIS
	ANALYTICAL STUDY OF THE RESINS AND DISCUSSION
	ANALYTICAL STUDY OF THE RESINS AND DISCUSSION

Fig 58 Comparison of B. bipinnata and B. stenophylla chromatograms. Identification

	ANALYTICAL STUDY OF THE RESINS AND DISCUSSION
	ANALYTICAL STUDY OF THE RESINS AND DISCUSSION
	B. stenophylla, type 1 sample 72 a2
	0.40
	0.20
	0.00
	0.00	5.00	10.00	15.00	20.00	25.00		30.00	35.00
				Minutes
	B. stenophylla , type 2 sample 72d2
	1.20
	1.00
	0.80
	0.60
	0.40
	0.20
	0.00
	0.00	5.00	10.00	15.00	20.00	25.00		30.00
				Minutes
	0.80
	0.60
	0.40						1
	0.20							2	3	4	5	6 7,8	9
	0.00
	0.00	5.00	10.00	15.00	20.00	25.00			30.00	35.00
				Minutes
	B. stenophylla, sample 72c2 sample
	1.50
	1.00
	0.50					T3	1
							2	3 5 4	6 7,8
	0.00
	0.00	5.00	10.00	15.00	20.00	25.00			30.00	35.00
				Minutes
									of triterpenic compounds
	is as follows: 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
	It is important to note that for B. stenophylla two lots can be distinguished: one with the presence
	of B4 and another one without it. Here in figure 59 we present a chromatogram of each type of
	B. stenophylla resin.
	PAOLA LUCERO								99

B. bipinnata, sample 11b2 sample

, lot 1 sample 47a1 B. laxiflora, lot 2 sample 47b2 B. laxiflora, lot 3 sample 47e1 * B. laxiflora, lot 4 sample 47d1 B. laxiflora, lot 5 sample 47c1 B. laxiflora, lot 6 sample 47f1 Fig 62 The six different molecular profiles for B. laxiflora

	ANALYTICAL STUDY OF THE RESINS AND DISCUSSION
	ANALYTICAL STUDY OF THE RESINS AND DISCUSSION
	1.20											0.80
	1.00
	0.80											0.60
	0.60											0.40					1
	0.20 0.40							1	2 3			0.20
	0.00											0.00
	0.00	5.00	10.00	Minutes 15.00 20.00	25.00	30.00	35.00	0.00	5.00	10.00	Minutes 15.00 20.00	25.00	30.00	35.00
	1.00											1.00
	0.80											0.80
	0.60											0.60
	0.40											0.40
	0.20
	0.00											0.20
	0. 2.00	6.00	10.00	14.00	18.00	22.00	26.00	30.00	34.00	38.00	0.00
					Minutes						0.00	5.00	10.00	15.00	20.00	25.00	30.00
															Minutes
	1.50											1.00
	1.00
												0.50
	0.50																2
																	1
	0.00											0.00
	0.00	5.00	10.00	15.00	20.00	25.00	30.00	35.00	0.00	5.00	10.00	15.00	20.00	25.00	30.00	35.00
					Minutes									Minutes
	* Due to a change in the instrumentation mode of injection (from a manual to an automatic injector) times of
	retention were enlarged, therefore to analyze this sample an enlarged protocol described in the Materials and
	methods section was used. The gradient remains the same, but 5 extra minutes were added to the running time.
	PAOLA LUCERO														102

Colima L2 OAXC1 OAXC3 OAXC2 Fig 65 Chromatograms of some commercial samples from unknown botanical origin, presumably not within Bursera species

	ANALYTICAL STUDY OF THE RESINS AND DISCUSSION
	ANALYTICAL STUDY OF THE RESINS AND DISCUSSION
	0.80
	0.60					1.50
	0.40					1.00
	0.20					0.50
	0.00					0.00
	0.00	10.00	20.00	30.00	35.	0.00	10.00	20.00	30.00	35
			Minutes					Minutes
	1.00					2.00
	0.80					1.50
	0.60					1.00
	0.40
	0.20					0.50
	0.00					0.00
	0.00	10.00	20.00	30.00	35	0.00	10.00	20.00	30.00	35
			Minutes					Minutes
	1.00
	0.50
	1.00
	0.00 0.80
	0.00 0.60	10.00	20.00	30.00	35
			Minutes
	0.40
	0.20
	0.00
	0.00	5.00	10.00	15.00	20.00	25.00	30.00	35.00	40.00	45.00
					Minutes
	PAOLA LUCERO								105

Table 20 TMS structure of the triterpenic standard molecules studied by GC-MS. O: Oleanane, U:Ursane, L: Lupane

 20

Two gradients of temperature were used for the study of molecular composition of copals, one for the study of global resins and one for the study of triterpenoids. In table 21 are reported the retentions times under both gradients (protocols are described in the materials and methods

Table 21 Time of retention of standard molecules under the designed GC-MS gradients

 21

Table 23 Recapitulative of botanical origin for archaeological and commercial samples studied

 23

Table 24 HPLC Gradient 1, employed for the study of the terpenic compounds in fresh resins

 24 For the study of archeological resins gradient 2 was used (table25).

	Time (min)	0	10	15	45
	% Metanol	85	95	100	100
	% H 2 O-TFA	15	5	0	0
	Flow : 1 mL.min-1
	Temperature of the column = 35 °C

Table 25 HPLC Gradient 2 employed for the study of the terpenic compounds in archaeological resins

 25

Table 26 Gardient 1, GC-MS temperature gradient for the global analysis of resins

 26

	PAOLA LUCERO

Table 27 Gradient 2, optimal gradient for the analysis of triterpenoids GC-MS

 27

Table 31 Pictures of some commercial samples

 31

	Annex 1

Table 32 . Selected pictures of optical microscopic study of certified origin samples from B. bipinnata species

 32

	Annex 1

Table 33 . Selected pictures of optical microscopic study of certified origin samples from B. bipinnata species, and from archeological sample M1 Annex 1 193

 33

		Electronical microscopic study
	SAMPLE	PICTURE
	11b3
	(35 X)
	11c2
	(35 X)

Table 34 Selected pictures of electronic microscopic study of certified origin samples from B. Bipinnata species

 34

	Annex 1

Table 35 Selected pictures of electronic microscopic study of certified origin samples from B. bipinnata species

 35

	Annex 1

Table 36 Selected pictures of electronic microscopic study of certified origin samples from B. Bipinnata species Annex 1 196 Pictures of an example of light aged copal

 36

Table 37 Picture of Sidral sample before and after light ageing Sidral (Commercial sample ) Before light ageing After light ageing Annex 1 197 Differences between B. Bipinata and B. Stenophylla

	Bursera bipinnata
	It has bipinnate to tripinnate leaves. The amount of
	leaflets and foliolulos (up to a hundred) per leaf,
	the distance between foliolulos is small and they
	are touching, and the end of the leaf, it has up to
	seven pairs of undivided leaflets.
	Bursera stenophylla
	Leaflets and foliolulos a does not exceed 10. The
	distance between leaflets and foliolulos is larger
	than in B. bipinnata and seldom touched each
	other. At the end of each leaf there are few pairs (
	two or three) of leaflets which are not divided into
	foliolulos

Table 38 Botanical differences between B. bipinnata and B. stenophylla Comparative of Spectra for botanical certified resins

Table 39 FTIR bands of certified resins used in PCA FTIR Data & PCA

 39

	Bands	a	b	c	d	e	f	g	h	i	j	k
	TMT	3433.16 2945.54 1708.92 1642.92 1455.24 1380.95 1243.17 1067.94	881.16	663.83	546.08
	173	3432.43 2948.23 1709.12 1663.77 1454.84 1379.24 1244.42	1067.6	881.57	663.2	584.6
	51	3432.15 2951.18 1706.53 1665.63	1454.9 1379.86 1247.25 1068.26	883.19	660.04	556.12
	52	3432.88 2945.13 1706.64 1642.84 1456.01 1380.84	1243.8 1067.78	881.83	665.23	547.31
	84	3435.27 2945.64	1712.2 1642.36 1454.91 1380.44 1242.82 1068.19	881.23	663.22	546.23
	26	3417.97 2946.88 1708.81 1659.94 1454.48 1378.92 1244.88 1066.64	882.41	662.23	547.05
	Chichen	3430.63 2950.89 1707.32 1654.94 1458.53 1384.31 1249.94 1078.32	883.73	665.6	581.87

Table 40 FTIR bands of archaeological resins used in PCA

 40

	Annex 2

Table 41 LDA results of classification of FTIR data of certified resins FTIR Data of Selected Commercial Samples

 41

	& PCA

Table 42 FTIR data used on PCA on selected commercial sampl Annex 2 209 PCA results for selected commercial resins

 42

	2200	2250	2300	2350
		First Component

Second Component Results of FTIR-PCA data for certified + commercial resins Fig 117 PCA: First two components for certified resins plus commercial samples:◄ATZM1, ◄CHOB1, ◄ATZB1, ◄ IZUR1. Symbols for certified resins are the same than the ones used along Chapter 3 Principal Component Analysis (FTIR data) Variables: a, b, k, c, d, e, f, g, h, i, j Eigenanalysis of the Covariance Matrix

											Annex 2 Annex 2
	grandifolia k g -20.160 -7.504 -5.274 -15.998 2539.15 3.387 1.401 2.098 i -0.065 2.218 bipinnata bipinnata grandifolia grandifolia grandifolia 0.147 5.580 7.489 bipinnata stenophylla stenophylla stenophylla laxiflora laxiflora laxiflora Variables: a, b, c, d, e, f, g, h, i, j, k excelsa 455.42 j 13.84 1.86 7.27 12.48 f 13.101 -2.153 -3.631 -4.901 5.615 10.891 h 0.844 Linear Method for Response: c1 bipinnata 17.60 i 1.569 0.793 1.626 0.559 e 22.464 -2.372 -2.315 -4.951 6.339 k -7.993 -0.100 -12.664 -6.260 0.972 0.380 -0.781 1.740 22.912 bipinnata bipinnata 2335.26 4.170 2.15 bipinnata 38.20 bipinnata 1540.31 5.44 0.99 stenophylla 33.50 stenophylla 2551.20 stenophylla 5.59 stenophylla 1547.47 1.911 1.489 penicillata 779.46 penicillata 4814.36 penicillata 777.27 penicillata 2032.48 HPLC Data & PCA
	laxiflora penicillata h 7.289 i -7.555 j 0.105 k -3.062 -2.863 3.897 1583.71 794.18 3.631 1.505 1.933 1.047 0.988 -2.310 17.390 8.481 3.303 A4 A3 A2 A1	-3.896 -3.328 B1	B2	-7.455 -12.687 -5.209 -4.653 excelsa grandifolia 2506.12 528.74 excelsa 2607.09 grandifolia 12.79 excelsa 502.88 grandifolia 2615.86 excelsa 1827.71 grandifolia 4888.51 B3 B4 B5 B6	B7	B8
	Group bipinnata excelsa grandifolia laxiflora penicillata stenophylla stenophylla 0.00 Variable stenophylla j -86.564 2.195 4.132 4.455 -16.608 -14.602 -1.977 laxiflora 1597.83 laxiflora 4587.84 laxiflora 1643.38 laxiflora 18.37 51 0.0475 0 0.0179 0.0146 0 0 0 0.0238 0	0 0.3021 0.0681
	Eigenvalue 2104.4 835.4 315.3 178.5 130.3 Proportion 0.565 0.224 0.085 0.048 0.035 0.020 0.014 0.003 0.003 75.4 53.6 12.9 9.6 Cumulative 0.565 0.790 0.874 0.922 0.957 0.977 0.992 0.995 0.998 Count 8 4 6 10 6 Summary of classification Linear Discriminant Function for Groups bipinnata excelsa grandifolia laxiflora penicillata stenophylla 8 a 7.804 b 4.282 c 4.816 d 4.100 e 1.026 k 2.429 -2.265 -0.516 -4.512 2.273 -1.732 4.924 h 8.462 i 3.910 2.645 j 4.519 8.119 52.904 k -0.884 0.036 -2.646 4.403 penicillata 748.58 penicillata 4530.65 penicillata 722.19 penicillata 1981.14 0.0191 0.0031 0.0054 0.1141 0 0 0 0.0479 0 0 0.0506 0.0336 52 Covariance matrix for Group stenophylla a b c d e f g h i j k a 60.908 stenophylla 29.32 8** bipinnata stenophylla stenophylla bipinnata 17.59 excelsa 457.97 grandifolia 2491.42 stenophylla 2374.99 penicillata penicillata penicillata bipinnata 794.74 excelsa 1444.53 grandifolia 4908.85 stenophylla 11.70 stenophylla stenophylla stenophylla bipinnata 45.52 excelsa 424.98 grandifolia 2328.96 stenophylla 1522.13 laxiflora laxiflora laxiflora TMT 0.266 0 0 0.0125 0 0 0 0.0552 0 0 0.0292 0.0187 bipinnata 1522.43 Chichén excelsa 1718.17 grandifolia 4766.76 Itzá 0.0905 0.0318 0.0227 0.0211 0.0201 0.0878 0 0.0184 0.0373 0 0.0522 0.0136
	Eigenvalue Proportion 0.002 0.000 6.5 1.4 Cumulative 1.000 1.000 Constant -3620271 -3644189 f 1.063 b -6.673 18.335 26 0.0792 0.0069 0.0048 0.0404 -3544947 True Group -3600516 0 0 Put into Group bipinnata excelsa grandifolia laxiflora penicillata -3641690 laxiflora 1603.47 -3623754 laxiflora 2258.01 laxiflora 1601.17 laxiflora 9.23 0 0.0169 0 0 0.0203 0.0469 bipinnata 6 0 0 0 0 excelsa 0 4 0 0 0 a -98 -99 -95 -99 -100 -98 b 752 754 748 750 750 752 c 659 662 648 657 663 659 g 3.497 h 0.179 i 2.094 Covariance matrix for Group grandifolia c -15.310 17.350 23.195 d 11.273 -6.480 -12.045 16.809 e 3.530 -2.681 -4.618 2.582 1.052 penicillata 794.66 stenophylla 8.80 excelsa excelsa excelsa bipinnata 488.64 penicillata 6.66 stenophylla 829.84 penicillata penicillata penicillata bipinnata 751.86 penicillata 817.33 stenophylla 24.44 stenophylla stenophylla stenophylla bipinnata 37.42 penicillata 1926.17 84 0.0298 0 0 0.1056 0 0 0 0 0.0247 0 0.0102 0.0526 stenophylla 1504.00 laxiflora laxiflora laxiflora bipinnata 1585.27 173 0.0723 0.0061 0 0.0065 0 0 0 0.002 0 0 0 0
	grandifolia d j a f -2.005		-26 28.46 b -1.155	0 -0.535 -27 c		0 -0.402 d	-33 0.018 e	6	-19 f 1.129	0	0 h i j k -15 excelsa g 4.27 excelsa 1453.89 excelsa 484.04 excelsa 1794.67	-26
	laxiflora e k a 132.085 g 10.622 -11.078 -16.558 0 1161 1162 6.230		0 8.783	1141 3.481	0	10 grandifolia 2785.81 0 1164 0.342 1163 12.231 grandifolia 4787.86 grandifolia 2652.67 grandifolia 4842.02	1162
	Variable penicillata f b 6.358 h -0.419		PC1 1102 0.415 -0.048		PC2 0.235 0 1110	PC3 -0.560 0	PC4 1087 -0.075 PC5 0	PC6 0.050 1100	PC7 1104 -0.202 laxiflora 1837.11 PC8 0 6 laxiflora 2147.82 laxiflora 1605.34 laxiflora 15.39	1103
	a stenophylla g c 9.045 i 6.972	0.155 -0.151 -36 0.889 10.306 2 -40 0.177 -0.130 0 -36 -5.157 -9.013 7.247 2.020 0.095 0	0.916 -0.216 -0.033 0 0 -35 -35 -0.046 6.723 penicillata 1354.42 penicillata 4.68 penicillata 851.42 penicillata 2033.01	-35
	b Total N h Pooled Covariance Matrix 0.035 0.097 -23 d 44.268 2.307 1.556 16.435 0.002 8 4 -22 j 22.551 57.362 71.613 -86.198 -12.638 -12.301 -51.306 0.008 0.062 -0.028 0.080 -0.011 6 10 6 -22 -22 -24 stenophylla 455.27 stenophylla 790.97 stenophylla 10.23 stenophylla 1565.24	-23
	k N correct i e 11.696 k 20.285 -19.831 -29.402 0.552 0.269 0.151 -0.000 6 4 900 905 909 0.232 -0.711 3.816 2.359 16.241 6.071 0.189 -0.161 -0.043 6 10 869 896 0.483 21.667 excelsa excelsa excelsa bipinnata penicillata penicillata penicillata bipinnata stenophylla stenophylla stenophylla bipinnata	0.256 6 485.64 699.40 27.57	900
	c Proportion j a f 14.934 h 0.032		0.481 -0.124 -0.603 0.750 1.000 45 46 b c 0.479 -1.731 4.682 d	0.466 1.444 1.000 0.253 45 e 4.619 f	0.094 1.000 45	0.133 -0.236 1.000 46 g h i j k excelsa 13.91 excelsa 1298.42 excelsa 498.92	45
	d k a 94.458 g -0.315 i -0.227		0.195 0.115 570 4.386	0.123 -0.219 -0.173 -1.110 0.152 0.005 579 569	0.119 -0.023 -0.093 575 572 2.013 2.571 grandifolia 2929.44 0.354 grandifolia 4647.36 grandifolia 2574.56	570
	e b -3.644 11.146 0.276 h -4.742 -0.228 j 1.285 -36.650 809.708 0.195 0.196 -0.237 -1.747 -0.602 -0.018 0.002 0.146 -0.032 -0.128 0.586 laxiflora 1749.18 0.357 laxiflora 1907.58 laxiflora 1540.34
	f Put into Group stenophylla -0.055 -0.057 -0.117 c -0.847 3.504 12.228 i -1.849 0.018 1.382 -0.596 -0.405 -0.844 -0.146 0.318 -0.497 0.239 k -0.351 11.888 -96.000 38.810 penicillata 1420.00 0.457 0.596 penicillata 10.71 penicillata 809.22
	g bipinnata d 12.223 j 55.736	-0.264 Pooled 0.090 4.623		0.281 -1.951 -8.361 25.312 -0.122 -0.053 -2.916 0.368 0.274 0.507 0.142 0 Means for Group 10.349 stenophylla 0.576 -0.144 449.81 stenophylla 736.19 stenophylla 5.63
	h excelsa Variable e 5.557 -3.309 -0.059 -0.703 -0.062 -0.293 0 Mean bipinnata excelsa grandifolia laxiflora penicillata 0.474 -0.131 0.203 0.352 -2.196 0.691 3.110 k -1.222 -0.085 -0.258 -0.443 -0.017 -0.139 -0.099 excelsa excelsa excelsa bipinnata 514.98 penicillata penicillata penicillata bipinnata 827.24 laxiflora laxiflora laxiflora bipinnata 1650.38
	i grandifolia -0.064 a 3428.3 f 7.541 -1.823 h 0.316 Summary of Classified Observations 0.469 -0.526 -0.618 0 3436.7 3422.7 -1.437 1.240 1.191	0.105 3417.0 3.480	0.161 3418.3 0.255 excelsa excelsa 1514.47 0.100 3436.7 9.78 excelsa 1809.04
	j laxiflora b g -8.709 -1.211 -0.495 2946.2 i 0.032 0.312		0.176 -0.300 0 2945.3 2948.3 0.349 -3.711 -0.318 0.566 -0.199 15.811 0.333 0.062 -0.505 2939.2 2950.0 grandifolia 2672.25 0.339 2946.3 grandifolia 5002.92 grandifolia 5061.22
	penicillata c 1701.8 h 11.475 0.034 j -3.879 -1.838 155.639 0 1711.5 2.574		1705.3 3.720	1635.4 0.022 -0.396	1705.9 3.161 laxiflora 1915.41 1732.1 Squared laxiflora 2031.99 laxiflora 15.58
	Variable stenophylla d 1643.9 PC9 i 1.287 -1.204 k 0.024 -0.017		PC10 1640.3 8 -2.008 -0.404	PC11 1634.2 2.387 0.022	1609.3 0.603 -0.110	1636.1 1.488 penicillata 1372.92 1704.5 Distanc penicillata 5.43 penicillata 2042.74
	a Total N e j -0.179 15.841 0.093 -0.032 1450.8 1455.4 8 15.176 -13.755 -6.490 -4.939 -8.496 0.007 1454.8 1424.7 1453.2 Observation True Group Pred Group X-val Group stenophylla Group stenophylla stenophylla 1629.46 1457.6 Pred 485.32 862.89
	b N correct f k 0.622 -3.364 0.717 1375.0 1 penicillata penicillata penicillata 0.669 0.132 8 1381.1 1380.4 1337.7 -6.889 1.230 1.402 -0.780 bipinnata bipinnata bipinnata excelsa excelsa excelsa laxiflora laxiflora laxiflora	1379.5 5.218 bipinnata bipinnata bipinnata bipinnata 1598.09 1384.7 3.74 500.10 764.01
	k Proportion g h 42.485 Covariance matrix for Group laxiflora 0.426 -0.538 -0.007 1.000 1244.0 1239.9 1219.9	1255.4	1247.5 excelsa excelsa excelsa 1332.26 1246.1 516.72 5.83 excelsa 1860.00
	c h i	1.528	-0.135 1062.8 2.460		0.092 1045.6	0.010 1080.4	1074.7	1078.3 grandifolia 2445.71 1029.8 grandifolia 2673.31 grandifolia 4843.82 grandifolia 4599.83
	d N = 42 i j -0.150 -7.505 191.663 -0.372 0.225 N Correct = 40 0.765 876.65 882.31 885.36 a b c d	Proportion Correct = 0.952 907.05 837.87 888.02 e f g h i j k laxiflora 1675.11 laxiflora 1869.86 laxiflora 1984.92 laxiflora 15.49
	e j k a 149.361 1.200	-0.320 665.16 1.635 -19.018 0.431 -0.627 664.27 676.57 11.470	661.24	685.86 penicillata penicillata 1427.94 661.88 777.04 penicillata 3.03 penicillata 2176.46
	f g k b -16.532 Summary of Classification with Cross-validation 0.077 -0.056 -0.038 -0.073 -0.063 -0.008 568.21 545.97 584.94 612.59 26.370 c -5.826 1.392 7.737 2 bipinnata bipinnata bipinnata grandifolia grandifolia grandifolia penicillata penicillata penicillata laxiflora laxiflora laxiflora	584.44 stenophylla stenophylla stenophylla stenophylla 1581.25 540.07 25.45 465.05 799.07 bipinnata 3.84 bipinnata 2525.94 bipinnata 745.12 bipinnata 1606.25
	h Covariance matrix for Group bipinnata 0.065 -0.021 -0.036 d 9.009 1.444 -2.090 12.883	excelsa excelsa 2766.63 499.58 excelsa 1303.32 excelsa 1880.00
	i Variable stenophylla -0.066 -0.015 -0.005 e 5.414 -10.294 -2.441 0.127	7.721	True Group grandifolia 2582.15 grandifolia 5.63 grandifolia 4808.30 grandifolia 4879.66
	j Put into Group bipinnata excelsa grandifolia laxiflora penicillata stenophylla 0.129 -0.100 -0.020 a 3437.2 a b c d e f g h i j k f 19.608 -5.446 -1.830 4.191 1.742 6.252 laxiflora 1635.64 laxiflora 4922.13 laxiflora 1976.96 laxiflora 6.67
	bipinnata b a 60.825 g -17.630			6 4.503 -0.488 -1.161 0 2946.3	0 -0.184 -2.462	0 3.376 penicillata penicillata 4816.86 0 714.84 penicillata 4.14 penicillata 2033.21
	excelsa c b 0.827 h 36.899			0 -7.002 -5.075 10.530 4 1712.2 0.549	2.967	0	0 8.183 -5.084 stenophylla stenophylla 2561.58 0 26.44 stenophylla 780.78 stenophylla 1595.37
	grandifolia d c 12.568 i 2.123 3**	0 7.383 -1.558 -4.087 1642.6 1.110 bipinnata stenophylla stenophylla 0 6 3.157 2.357 2.125 grandifolia grandifolia grandifolia stenophylla stenophylla stenophylla laxiflora laxiflora laxiflora	0 0.312 bipinnata bipinnata 2664.15 0 18.14 bipinnata 28.45 bipinnata 1871.09
	laxiflora e d 2.340 -0.081 -0.330 0 1455.8 j -27.064 14.710 5.687 -4.343 -10.401 -4.619 0 0 0.991	10 3.441 excelsa excelsa 2912.64 0 470.23 excelsa 420.85 excelsa 2138.12
	penicillata f e 0.057 -0.295 -1.082 0 1381.3 k -7.805 2.229 2.064 -2.858 0 0.524	0 -0.294 -2.450 0.464	0 1.210 grandifolia 2504.77 6 grandifolia 7.14 grandifolia 2632.25 grandifolia 5143.70
	stenophylla g f -0.562 -0.605 -1.464 2 1245.8 h 18.326		0 0.257	0.420	0 0.751	0 laxiflora 1464.65 0 laxiflora 4986.62 laxiflora 1629.72 laxiflora 20.87
	Total N h g -24.440 i 5.104			1068.0 1.979 3.653	8 0.839 -2.200 -1.916 -2.854 52.951 4 6 10 penicillata penicillata 5063.21 6 805.26 penicillata 822.12 penicillata 2272.57
	N correct i h 11.937 j -10.659			6 8.048 15.584 883.79 -3.887 29.545		4 3.000 -1.263 -9.359 27.973 6 10 stenophylla stenophylla 2703.93 6 12.09 stenophylla 12.05 stenophylla 1862.62
	Proportion j i -0.373 k -3.861	4		0.750 639.84 0.125 1.235 -0.627 -0.443 -0.309 1.000 1.000 -0.918 0.126 3.027 bipinnata bipinnata bipinnata grandifolia grandifolia grandifolia stenophylla stenophylla stenophylla laxiflora laxiflora laxiflora	1.000 2.637 bipinnata bipinnata 2585.04 1.000 3.50 bipinnata 13.94 bipinnata 1545.88	1.000
	k j	7.185			549.64 0.794	2.928		0.631 -0.252 -0.816	6.037	excelsa excelsa 2837.89 519.39 excelsa 492.16 excelsa 1777.10
	N = 42 k -1.511			N Correct = 40 0.767 0.768 0.479 -0.138 -0.774 Proportion Correct = 0.952 2.131 grandifolia 2570.24 grandifolia 5.78 grandifolia 2502.83 grandifolia 4649.66
	h 190.448 Covariance matrix for Group penicillata	laxiflora 1531.40 laxiflora 4982.65 laxiflora 1603.80 laxiflora 5.70
	i	-0.127			0.629						penicillata penicillata 4969.31 716.92 penicillata 824.21 penicillata 2096.74
	Squared Distance Between Groups j 12.405 0.135 3.452 a b c	d	e	f	g stenophylla h i j k 30.26 stenophylla 2614.24 stenophylla 5.34 stenophylla 1533.61
	k a	Pooled 14.032 -0.258 7.664 5 bipinnata 1.106 grandifolia grandifolia grandifolia StDev for Group 1.963 bipinnata bipinnata stenophylla stenophylla stenophylla laxiflora laxiflora laxiflora	bipinnata bipinnata 2443.60 23.47 bipinnata 19.15 bipinnata 1639.98
	Variable b 3.273		bipinnata excelsa grandifolia laxiflora penicillata StDev bipinnata excelsa grandifolia laxiflora penicillata 4.367 excelsa 411.93 excelsa 2685.56 excelsa 420.84 excelsa 1879.67
	bipinnata a c -0.345 -5.026 9.719	0.00 20.008 488.90 7.799 12.979	2504.58 11.493 1600.45 12.221 grandifolia 2527.60 757.95 2.768 grandifolia 4.69 grandifolia 2566.75 grandifolia 4889.19
	excelsa b Covariance matrix for Group excelsa 488.90 0.00 3.339 0.741 1.615 d 3.636 1.628 3.281 2.994	2756.76	1834.44 0.644	1385.37 laxiflora 1652.14 5.135 2.090 laxiflora 4793.93 laxiflora 1580.08 laxiflora 13.85
	grandifolia c e 0.070	2504.58 2756.76 3.497 2.717 0.062 -1.339 -0.448 1.286	0.00 0.208	4840.90 3.210 penicillata 4827.41 2.782 penicillata 4811.34 4.473 806.80 penicillata 811.86 penicillata 1984.07
	laxiflora d f -0.198 -0.045 1600.45 1834.44 3.217 0.995 a b c -0.343 -0.152 3.409 d	4840.90 e 0.011	4.054 0.017	0.00 f stenophylla 2045.44 3.589 g h i j k 1.730 54.89 stenophylla 2470.23 stenophylla 7.18 stenophylla 1620.14
	penicillata e a 168.451 g 0.785 6	757.95 1385.37 0.681 0.301 1.763 -0.449 0.359 -0.020 4827.41 2.518 bipinnata bipinnata bipinnata 2045.44 1.536 0.011 0.190 2.779 bipinnata 0.00 grandifolia grandifolia grandifolia bipinnata 2514.73 0.456 7.48 stenophylla stenophylla stenophylla bipinnata 12.74 laxiflora laxiflora laxiflora bipinnata 1569.03
	stenophylla f g b 3.463 c -0.792 h 0.446 -0.459 1.865 3.976 2.609 17.60 1.696 1.654 455.42 0.866 7.277 3.577 1.071 -0.371 -0.055 2539.15 1583.71 3.300 2.149 4.787 1.603 0.057 2.500 1.837 i 2.586 0.258 1.249 0.852 0.290 -0.150 -0.014 excelsa 794.18 grandifolia 2488.51 0.132 0.436 586.53 excelsa 2772.01 grandifolia 5.24 excelsa 480.83 grandifolia 2521.50 excelsa 1784.18 grandifolia 4812.66 Linear Discriminant Analysis: FTIR data stenophylla h 6.518 13.800 2.909 0.562 4.281 0.918 d 8.044 5.500 3.672 11.624 j 1.996 0.218 0.555 0.587 0.126 -0.070 0.112 laxiflora 1723.26 laxiflora 4813.48 laxiflora 1547.97 laxiflora 3.09
											211 212

Table 44 HPLC data of peaks corresponding to non triterpenic compounds in botanical certified resins and archaeological resins used in PCA Annex 3 226 HPLC Data & PCA T3 Epi lupeol lupeol Epi βamyrina lupenone Epi αamyrin β amyrin αamyrin/β amyrona α amyrone

 44

	T1

Epi βamyrina lupenone Epi αamyrin β amyrin αamyrin/β amyrona α amyrone

										T1
	72a2	0.0271	0.2145 0.0141	0.0139	0.0838	0.02599	0.08323	0.1064	0.0113 0.0113
	72b1	0.0216	0.226	0.022	0.0184	0.0778	0.03036	0.10866	0.0688	0.0263 0.0263
	72b2	0.0315	0.0193 0.0112	0.0136	0.0819	0.02331	0.09656	0.0745	0.0169 0.0169
	72c1	0.0013	0.272 0.0192	0.0041	0.09463	0.02175	0.12298	0.0986	0.0276 0.0072
	72d1	0.0469	0.1998	0.025	0.0203	0.08103	0.03186	0.10288	0.0893	0.0203 0.0203
	72d2	0.042	0.1926 0.0299	0.0134	0.07321	0.0272	0.08268	0.0857	0.0212 0.0107
	72c2	0.0709	0.1926 0.0207	0.0137	0.05241	0.01822	0.05702	0.0633	0.0199 0.0089
	51	0	0.1797 0.0199	0	0.06983	0.02434	0.06022	0.1034	0.0168 0.0347
	52	0	0.3122 0.0314	0	0.09262	0.02435	0.05261	0.1528	0.0068 0.0041
	TMT	0	0.2878 0.0206	0	0.08422	0.01833	0.05112	0.1563	0	0
	Chichén Itzá	0	0.0328	0	0.0164	0.01759	0	0.14822	0.0255	0.1482 0.2321
	26	0	0.2978	0.022	0.0126	0.10526	0.02753	0.07452	0.1858	0.0043 0.0521
	84	0	0.3049 0.0215	0.0299	0.09856	0.02619	0.07091	0.1537	0.0036 0.0357
	173	0	0.3344 0.0224	0	0.12263	0.03345	0.09979	0.2032	0.0184 0.0458

Table 45 HPLC data of peaks corresponding to non triterpenic compounds in botanical certified resins and archaeological resins used in PCA Chromatograms of Commercial Samples

 45

	ATZB1 C= 1.75mg/mL
		0.40
	A U
		0.20
		0.00
		0.00	2.00	4.00	6.00	8.00	10.00	12.00	14.00	16.00	18.00	20.00	22.00	24.00	26.00	28.00	30.00	32.00	34.00
											Minutes
	ATZM1 C= 1.75mg/mL
		0.40
	A U	0.20
		0.00
		0.00	2.00	4.00	6.00	8.00	10.00	12.00	14.00	16.00	18.00	20.00	22.00	24.00	26.00	28.00	30.00	32.00	34.00
											Minutes
	CHOB1 C= 3 mg/mL
		0.40
		0.30
	A U	0.20
		0.10
		0.00
		0.00	2.00	4.00	6.00	8.00	10.00	12.00	14.00	16.00	18.00	20.00	22.00	24.00	26.00	28.00	30.00	32.00	34.00
											Minutes
	CHOB2 C= 3 mg/mL
		0.40
		0.30
	A U	0.20
		0.10
		0.00
		0.00	2.00	4.00	6.00	8.00	10.00	12.00	14.00	16.00	18.00	20.00	22.00	24.00	26.00	28.00	30.00	32.00	34.00
											Minutes

Samples analyzed after the installation of the automatic injector under HPLC gradient number 2

	CHOR1 C= 12.5mg/ mL
	1.50
	1.00
	0.50
	0.00
	0.00	10.00	20.00	30.00	40.00	50.00
				Minutes
	IZUR1 C= 6mg/mL
	3.00
	2.00
	1.00
	0.00
	0.00	10.00	20.00	30.00	40.00	50.00
				Minutes

Fig 118 Chromatograms of commercial samples Principal Component Analysis for HPLC data Variables: A4, A3, A2, A1, B1, B2, B3, B4, B5, B6, B7, B8, etc Eigenanalysis of the Correlation Matrix

							Annex 3 Annex 3 Annex 3 Annex 3 Annex 3
	β amyrin B14 B11 E1 D1 Epi βamyrina A2 β amyrin		0.024 -0.046 -0.136 0.00000 0.000015 0.000062 -0.000015 -0.000077 0.000000 0.059 0.066 -0.119 -0.109 0.000000 0.000000 -0.001298 -0.001817 0.012074 -0.000143 0.000000
	αamyrin/β amyrona B15 B12 D1 T3 lupenone A1 αamyrin/β amyrona -0.000079 0.004 0.00000 0.072 -0.160 -0.055 -0.050 0.0116194 0.000000 0.000000 0.000000 0.000000 0.000000 0.164 -0.092 0.000000 -0.000052 0.000000 0.000167 0.005378 0.006174 -0.005193 0.024457 0.000000
	α amyrone Linear Discriminant Function for Groups 0.045 -0.028 -0.059 E1 0.00000 Pooled Covariance Matrix P1 0.0000000 T3 0.000000 0.000000 Epi lupeol 0.000000 0.001138 Epi αamyrin 0.000000 0.000000 B11 -0.003384 -0.004660 α amyrone 0.000023 0.000000	0.058 -0.141 -0.076 -0.041 0.000000 0.000000 0.000000 0.000000 0.000000 0.000276 -0.015401 0.041854
	T4 D1 P2 Epi lupeol lupeol β amyrin B12 T1		0.077 -0.006 -0.241 -0.076 -0.065 0.00000 -0.0000000 0.000210 0.000385 0.000241 -0.000952 0.000000 0.028 0.006 0.000000 0.000075 0.000132 0.000000 -0.000030 0.000000 0.000032 -0.002047 -0.002089 -0.008551 -0.010732 -0.014267 0.000000 0.000000
	T3 B14 lupeol Epi βamyrina αamyrin/β amyrona P1 P2	bipinnata excelsa laxiflora penicillata stenophylla 0.03825 -0.0000000 0.000020 0.000021 0.000012 -0.000075 0.000000 0.000000 0.000062 0.000000 0.000000 0.000000 0.000000 0.000000 0.000000 0.000000 0.000000 0.000000 0.000000
	Variable Constant Epi lupeol B15 Epi βamyrina lupenone α amyrone P2 B14		PC15 -16822 0.18452 PC16 -16070 A4 0.0000000 0.000011 0.000032 PC17 A3 0.000000 0.000289 -0.000009 0.000000 -0.000001 PC18 -15564 0.000021 -0.000143 0.000000 PC19 PC20 PC21 -21660 -15525 A2 A1 0.000000 -0.000001 0.000000 0.000000 0.000000 0.000000 0.000000	B11
	A4 A4 lupeol A4 E1 lupenone Epi αamyrin T1 B14 B15		-0.186 20013 0.02028 0.084 -0.149 -0.044 -0.033 19668 20062 20707 0.002 0.0005946 0.0000000 0.000034 0.000066 0.000058 -0.000471 0.000000 0.029 19466 0.000000 0.000109 0.000000 0.000000 0.000000 0.000000 0.000000 0.000000 0.000000 0.000000 0.000000 0.000000
	A3 A3 Epi βamyrina A3 D1 Epi αamyrin β amyrin T1 B15 E1		-0.105 -0.025 34892 35207 0.057 0.01453 0.0004495 0.0010995 0.0004196 0.000020 0.000040 0.000000 0.000165 0.000000 0.000000 0.000000	0.017 -0.021 -0.041 -0.008 34555 39224 33328 0.000025 -0.000136 0.000000 0.000000 0.000000 0.000000
	Eigenvalue 6.4080 3.2056 2.6222 1.9800 1.3123 1.2851 0.9155 0.8665 A2 -0.243 -0.133 -0.173 0.062 0.056 -0.042 0.014 A2 32138 31765 31468 35532 30812 lupenone 0.07654 A2 -0.0004010 -0.0005057 0.0038624 T3 -0.0000000 β amyrin 0.000003 -0.000002 0.000014 -0.000830 0.000000 αamyrin/β amyrona 0.000000 0.000311 E1 0.000000 0.000000 0.000000 0.000000 0.000000 D1
	Proportion A1 A1 Epi αamyrin A1 Epi lupeol αamyrin/β amyrona 0.291 α amyrone D1 T3	0.146 0.308 33890 0.02544 0.119 0.010 -0.081 0.090 33107 0.0017711 0.0025658 -0.0016662 0.060 0.051 -0.001 0.058 32557 37709 0.078 0.042 0.0138005 0.0002604 0.000026 0.000039 0.000079 -0.000168 0.000000 0.039 0.101 32404 0.000000 -0.000027 Epi -0.000434 -0.000334 0.000264 -0.001777 -0.000210 0.000000
	Cumulative B1 B11 β amyrin B11 lupeol α amyrone T1 T3 Epi lupeol	0.291	0.437 0.025 32325 0.09463 0.556 0.030 -0.011 0.646 31830 -0.0010635 -0.0026780 -0.0003280 -0.000016 -0.000057 -0.000027 -0.000300 0.000000 0.706 0.764 0.806 0.845 0.042 -0.001 0.001 0.122 31302 35886 30936 0.0000868 -0.0101369 0.0225355 0.000000 0.000000 B12 P1 P2 B14 B15 E1 D1 T3 lupeol 0.000000 0.000000 0.000000 0.000000 0.000000 0.000000 0.001816
	B2 B12 αamyrin/β amyrona B12 Epi βamyrina T1 lupeol A4 Epi lupeol lupeol	-0.180 -0.109 -0.080 32104 31595 0.08098 -0.0006434 -0.0006566 -0.0026873 -0.0033731 -0.0044839 0.022 0.033 -0.016 0.058 31144 35615 30739 -0.0000343 0.000000 0.000000 0.000000 0.000000 0.000000 -0.000531 -0.000454 0.003202 -0.002078 -0.003626 0.000000 -0.000113
	Eigenvalue 0.8082 0.7062 0.6103 0.3526 0.2973 0.2435 0.1585 0.1040 B3 -0.009 0.087 -0.077 0.157 0.177 0.511 0.435 P1 28353 29885 28949 39002 26533 α amyrone 0.02133 P1 0.0000000 0.0000000 0.0000000 -0.0001190 0.0000097 lupenone -0.0000039 P2 0.007073 Epi βamyrina A3 lupeol -0.000286 -0.000324 0.000410 -0.000569 0.000334 Epi βamyrina 0.000000 0.000142
	Proportion B4 P2 T1 P2 Epi αamyrin B14 lupenone A2 Epi βamyrina lupenone	0.037	0.032 0.009 -0.087 0.028 31079 30164 0.141 -0.128 -0.048 0.016 0.014 0.011 29691 33595 0.052 0.007 0.01297 -0.0004561 -0.0001598 -0.0000639 -0.0007972 -0.0000443 0.005 0.055 29777 -0.0000263 -0.000035 0.000083 -0.000071 -0.000086 0.000345 -0.000232 -0.000140 0.000000 0.000130
	Cumulative B5 B14 B14 β amyrin B15 Epi αamyrin A1 lupenone Epi αamyrin	0.882	0.914 -0.019 44385 0.0000049 0.154 -0.016 -0.369 -0.055 -0.555 -0.231 0.942 0.958 0.971 0.982 0.990 0.994 43042 41775 56848 41771 0.0000104 -0.0000093 0.0001722 -0.0002142 -0.0000450 0.000000 0.000000 0.000000 -0.000036 0.000009 0.000168 -0.000139 -0.000256 0.000000 0.000056
	B6 B15 B15 αamyrin/β amyrona -0.0000249 -0.444 -0.002 -0.173 67277 76938 0.0000000 0.0000088 -0.0000000 0.058 -0.056 73794 76835 0.130 -0.076 62552 0.0001763 -0.0003108 E1 -0.000131 0.000103 0.000000 0.000168 β amyrin B11 Epi αamyrin -0.000037 -0.000049 0.000163 -0.000083 -0.000103 β amyrin 0.000000 0.000032
	Eigenvalue 0.0634 0.0298 0.0162 0.0111 0.0030 0.0007 B7 -0.223 -0.106 0.036 0.029 0.101 -0.044 E1 24202 22004 22115 19525 StDev for Group E1 0.0000039 0.0000159 -0.0000039 -0.0000198 α amyrone 0.0000073 D1 0.000000 0.000000 0.000000 0.000000 0.000000 0.046 23505 0.0000000 αamyrin/β amyrona 0.000237 B12 0.000000 β amyrin 0.000022 0.000094 -0.000005 0.000073 -0.000142 αamyrin/β amyrona 0.000000 0.000036
	Proportion B8 D1 Variable D1 T1 T3 α amyrone P1 αamyrin/β amyrona 0.003 α amyrone	0.001 0.493 34560 Pooled StDev bipinnata 0.001 0.001 0.387 -0.057 34580 -0.0001364 -0.0001085 -0.0000000 0.000000 0.000000 -0.000005 0.000032 0.000000 0.000000 0.000024 0.000034 0.000000 0.000002	0.000 0.040 -0.072 0.000 35044 37742 0.049 -0.039 33103 excelsa laxiflora penicillata 0.0000830 -0.0004069 -0.0003345 0.000000 0.000000 0.000000 0.000012 0.000076 -0.000156
	Cumulative T3 T3 A4 T3 P1 Epi lupeol T1 P2 α amyrone T1	0.997	0.999 0.309 -0.116 0.999 2890 -493 0.080 1.000 0.02438 0.00965 0.0000594 0.0000243 -0.001982 -0.000046 0.000000 0.000000 0.000000 0.000000 0.000008 0.000007 -0.000049 1.000 0.005 -663 0.00000 0.0000082 0.152 -0.081 1.000 -1455 0.03576 0.0003065 -0.0003037 0.019 4154 0.00000 0.000000 0.000021 0.000000 0.000046 -0.000121 0.000000 0.000000
	Epi lupeol Epi lupeol A3 Epi lupeol P2 lupeol T1 B14 T1 lupeol		-0.113 28255 0.03316 0.091 27326 0.716 -0.226 26553 0.02425 0.05666 0.0001145 -0.0000411 0.0010465 -0.0008726 -0.0018908 0.030 0.114 0.162 30939 27054 0.04563 0.00000 -0.000140 0.000006 0.000000 0.000001 0.000000 0.000000 0.000000 0.000000 0.000000 0.000000 0.000000 0.000000
	lupeol lupeol A2 lupeol B14 Epi βamyrina B15 P2 Epi βamyrina		0.080 -0.007 17478 16931 0.024 0.06215 0.01143 -0.0000660 -0.0000159 -0.000054 -0.000002 0.000000 0.000000 0.000000	0.006 16733 0.00000 0.0001252 0.076 -0.063 18599 0.10988 0.0000399 -0.0004346 0.051 16968 0.00000 0.000000 0.000000 0.000000
	Variable Epi βamyrina Epi βamyrina A1 Epi βamyrina B15 lupenone E1 B14 lupenone		PC1 -0.241 -0.120 PC2 22028 22316 0.043 PC3 0.1175 0.0776 -0.0000203 -0.0000137 -0.000302 -0.000031 0.000000 0.000000 0.000000 0.000000	PC4 0.033 20346 0.1896 0.0001125 -0.0001549 -0.0000970 PC5 PC6 PC7 0.088 -0.039 0.007 38010 19361 0.1564 0.0731 0.000000 -0.000024 0.000000
	A4 lupenone lupenone B11 lupenone E1 Epi αamyrin D1 B15 Epi αamyrin		0.195 -0.176 -0.005 -0.219 0.010 0.038 -0.262 0.258 -0.164 -0.489 0.027 -0.094 1399 -2927 -2211 -3716 0.1501 0.0000 0.3280 0.2046 -0.0000457 0.0000103 0.0000542 -0.0001968 -0.0003039 0.636 0.618 2820 0.0334 -0.000168 -0.000004 0.000000 0.000001 0.000000 0.000000 0.000000 0.000000 0.000000 0.000000
	A3 Epi Epi B12 Epi D1 β amyrin αamyrin αamyrin αamyrin Covariance matrix for Group excelsa -0.092 -0.088 -0.374 -0.334 -0.087 -0.007 0.056 -0.335 -0.235 -0.597 187044 187676 186754 205004 0.097 0.349 -0.199 0.062 177598 0.1078 0.0000 0.0000 0.1923 0.0000 -0.0000150 -0.0000062 0.0000522 -0.0001128 -0.0000478 0.0001782 0.000325 -0.000035 0.000000 -0.000029 0.000000 T3 0.000000 0.000000 E1 0.000000 0.000000 0.000000 0.000000 β amyrin
	A2 β amyrin β amyrin P1 β amyrin T3 αamyrin/β amyrona -0.000568 -0.000068 -0.209 -0.234 0.134 -0.109 0.618 -0.212 37079 36046 0.005681 0.000000 0.000000 0.096 -0.101 -0.344 -0.104 0.019 0.510 0.040 -0.162 35142 42496 35385 0.000000 0.015030 -0.0000177 0.0000283 -0.0000346 -0.0003848 -0.0001882 0.0000263 0.000000 -0.000096 0.000000 Epi lupeol 0.000000 0.000000 D1 0.000000 0.000000 0.000000 0.000000 0.000490 αamyrin/β amyrona 0.000009
	A1 αamyrin/β amyrona αamyrin/β amyrona P2 αamyrin/β amyrona -0.0000281 0.007 0.208 -0.481 -0.063 -0.177 0.211 -0.473 -0.116 0.333 0.298 38749 37353 36849 41103 0.136 -0.110 0.015 -0.404 37090 0.05569 0.08410 0.00000 0.00000 0.02027 0.0000071 0.0000185 -0.0002645 -0.0000559 Epi lupeol 0.0001873 α amyrone 0.000346 0.000004 0.000000 0.000001 0.000000 lupeol 0.000000 0.000000 T3 0.000000 0.000000 0.000000 0.000000 0.000000 α amyrone 0.000004 0.000006
	B1 α amyrone α amyrone B14 α amyrone lupeol T1 Epi βamyrina Epi lupeol T1		-0.320 0.036 77601 0.01072 0.002 0.086 78781 0.081 0.277 0.00913 0.0000176 -0.0000201 -0.0000303 -0.0001021 -0.0000439 0.072 0.100 -0.070 0.105 0.526 -0.400 -0.025 -0.121 77640 90278 74731 0.01603 0.00000 0.02236 -0.0000236 0.000000 0.000000 0.000000 0.000000 0.000000 A4 A3 A2 A1 B11 0.000000 0.000000 0.000000 0.000000 0.000000 0.000000 0.000558 0.000000 0.000000
	B2 T4 T1 B15 T1 Epi βamyrina lupeol A4 lupenone lupeol T1		-0.161 -0.166 0.217 -0.329 -0.184 -0.494 0.197 -0.020 -0.230 -0.093 -0.004 0.100 0.088 0.246 -23910 -26593 -26212 -32395 -19759 0.004693 0.000000 0.016029 0.000000 0.000000 -0.0000152 0.0000029 0.0000027 -0.0000272 -0.0000000 0.0000131 0.000011 0.000000 0.000000 0.000000 0.000000 0.000000 0.000000 0.000000 -0.000067
	B3 E1 P1 lupenone Epi βamyrina A3 Epi αamyrin Epi βamyrina		-0.068 0.006574 0.480 0.0000323 0.0000381 0.000006 0.000007 0.140 0.012964 0.000000 0.138 0.000000 0.003210 0.000000 0.000000 0.000000 0.000000 0.000000	0.173 0.000000 0.086 0.000000 0.000042 0.204 0.000000
	B4 Variable D1 P2 Epi αamyrin lupenone A2 β amyrin lupenone		0.201 PC22 0.01335 0.185 -0.103 0.00000 0.0000338 0.0031018 0.0000012 0.000021 0.000020 0.000000 0.000000 0.000000 0.000000 0.000000 0.000000	0.117 -0.403 -0.115 -0.381 0.01684 0.02213 0.00000 0.000100 0.000000 0.000000 0.000000 0.000063
	B5 A4 T3 B14 β amyrin Epi αamyrin A1 αamyrin/β amyrona Epi αamyrin	-0.070 0.134 0.01157 0.498 0.0000191 -0.0000202 0.104 0.00000 0.0000079 0.000009 0.000008 0.000000 0.008194 0.000000 0.000000 0.000000 0.000000	0.086 Means for Group 0.171 0.148 0.01843 0.00000 0.0001149 0.000032 0.000012 0.000000 0.035967 0.000000 0.000000 0.000004 0.150 0.00000
	B6 A3 Variable Epi lupeol B15 αamyrin/β amyrona β amyrin B11 α amyrone β amyrin Covariance matrix for Group penicillata -0.117 -0.009 -0.357 -0.217 0.110 Pooled Mean bipinnata excelsa laxiflora penicillata 0.312 0.223 -0.148 0.04514 0.03374 0.02725 0.04262 0.00776 -0.0000000 -0.0000000 0.0000220 0.0000220 0.0000075 0.000009 0.000019 0.000092 0.000023 0.000166 0.000000 -0.014158 0.000000 -0.062173 0.107592 0.000000 0.000000 0.000000 0.000000 0.000000 0.000000 0.000035
	B7 A2 A4 lupeol E1 α amyrone αamyrin/β amyrona B12 T1 αamyrin/β amyrona	0.081 -0.131 0.256 0.02994 0.02365 0.0000000 -0.0000337 0.190 -0.044 0.01815 0.00000 0.00328 0.02490 0.0000264 -0.0000000 0.144 0.559 -0.307 0.02554 0.00000 0.03799 0.01737 0.0000432 0.0000021 0.000024 0.000013 0.000107 0.000030 0.000038 0.000000 0.000000 0.000000 0.000000 0.000000 0.000000 0.000000 0.000000 0.000000 0.000000 0.000000 0.000024
	B8 A1 A3 Epi βamyrina D1 T1 α amyrone P1 P2 α amyrone		-0.198 -0.339 0.393 0.02670 0.006911 -0.0000000 -0.0000000 0.200 0.01928 0.07057 0.040 0.002550 0.000000 0.0000228 0.142 0.03337 0.099 -0.132 0.00000 0.004751 0.015392 0.0000228 -0.0000000 -0.0000000 -0.000000 0.000001 0.000008 -0.000002 0.000043 0.000000 0.000000 0.000000 0.000000 0.000000 0.000000 0.000000 0.000000 0.000000 0.000007
	T3 B1 A2 lupenone T3 T3 T1 P2 B14 T1		0.094 -0.014 -0.288 -0.252 0.601 0.06514 0.02458 0.00000 0.009397 0.010002 0.012904 -0.0000000 -0.0000001 0.0000249 0.200 -0.352 0.18000 0.005135 0.0000249 -0.0000000 0.082 0.00000 0.007689 0.000000 0.000000 0.000000 0.000000 0.000000 0.000000 0.000000 0.000000 0.000000 0.000000 0.000000 0.000000 0.000000 0.000000 0.000000 A4 A3 A2 A1 B11
	Epi lupeol B2 A1 Epi αamyrin Epi lupeol Epi lupeol T1 B14 B15 lupeol A4		0.318 -0.238 -0.020 0.311 0.23205 0.32666 0.33166 0.134 0.003754 0.003441 0.000000 -0.0000023 -0.0014620 -0.0000130 0.140 0.13638 0.054 0.003354 0.0000166 0.000000 0.000000 0.000102 0.000000 0.002057 -0.003626 0.116 0.23889 0.004970 0.0000054 0.001443
	lupeol B3 B11 β amyrin lupeol lupeol B15 E1 Epi βamyrina		-0.053 -0.080 -0.095 0.019 0.17896 0.00000 0.38575 0.011 0.01522 0.01287 0.00564 -0.0000102 -0.0001070 -0.0000386 0.622 -0.230 -0.288 0.35279 0.23035 0.00421 0.02646 0.0000021 0.0000003 0.000000 0.000102 0.000000 0.002057 -0.003626 0.000055 0.000023
	Epi βamyrina B4 B12 αamyrin/β amyrona Epi βamyrina Epi βamyrina E1 D1 lupenone	0.156 0.201 0.04173 0.259 0.01457 -0.0000099 0.0000105 -0.0000432 0.129 -0.003 0.182 -0.427 -0.214 0.00000 0.00000 0.13911 0.00000 0.01540 0.00000 0.00294 0.02554 0.0000000 0.0000001 Epi 0.000000 0.000000 0.000000 0.000000 0.000000 0.000002 0.000017 0.000026
	lupenone B5 P1 α amyrone lupenone lupenone D1 T3 Epi αamyrin		0.371 -0.108 -0.052 0.227 0.01506 0.00000 0.00000 0.132 0.004466 0.005673 0.000396 0.0000065 -0.0000537 -0.0000018 0.029 0.00000 0.059 -0.028 0.10042 0.002537 0.004771 0.0000172 -0.0000063 B12 P1 P2 B14 B15 E1 D1 T3 lupeol 0.000000 -0.000043 0.000000 0.001767 -0.003133 0.000340 0.000106 0.000010 0.000003 0.000011
	Epi B6 P2 T1 Epi Epi A4 T3 Epi lupeol αamyrin αamyrin αamyrin β amyrin		0.365 -0.039 -0.010 0.062 0.06204 0.13156 0.00000 0.167 0.003466 0.000000 0.000000 -0.0000018 -0.0000484 -0.0000118 0.144 -0.089 -0.057 0.00000 0.06019 0.000000 0.000000 0.0000000 0.0000004 0.0000141 0.000000 0.000025 0.000000 0.001987 -0.003543 0.000154 0.000743 0.000001 -0.000001 0.000010 -0.000002 0.000018
	β amyrin B7 B14 β amyrin β amyrin A3 Epi lupeol lupeol αamyrin/β amyrona	0.355 -0.002 0.110 0.03731 -0.0000061 0.0000824 -0.0000436 -0.0000018 -0.0000075 0.055 -0.175 -0.118 -0.078 -0.160 0.01599 0.02837 0.00000 0.20313 0.0000272 0.000000 0.001298 0.000000 0.004936 -0.008461 -0.000048 0.000599 0.000002 0.000001 0.000007 0.000000 0.000008
	αamyrin/β amyrona B8 B15 αamyrin/β amyrona αamyrin/β amyrona A2 lupeol Epi βamyrina α amyrone	0.343 -0.030 0.102 0.00284 0.0000465 -0.0001512 -0.0000125 0.067 0.204 0.112 0.00000 0.02837 0.00000 0.176 0.0000000 -0.0000248 0.156 0.00000 0.0000105 0.000000 0.001009 0.000000 0.003362 -0.005633 0.000000 0.000000 0.000049 0.000005 0.000007 0.000004 0.000002
	α amyrone T3 E1 Variable α amyrone α amyrone A1 Epi βamyrina lupenone T1		0.096 0.010 0.00353 0.217 stenophylla 0.0000015 0.0000643 -0.0000097 -0.0000003 0.314 -0.499 -0.007 -0.037 -0.064 0.01413 0.00000 0.00000 0.00000 0.0000004 0.0000025 0.000000 0.000000 0.000000 0.000000 0.000000 0.000237 0.000095 0.000000 0.000000 0.000000 0.000000 0.000000
	T4 Epi lupeol D1 A4 T1 T1 B11 lupenone Epi αamyrin T1		0.107 -0.007 0.255 0.01630 0.02903 -0.0000000 0.0000175 0.322 -0.532 0.00000 0.01997 0.0000000 0.017 0.04768 0.077 -0.090 0.00000 0.0000000 -0.0000000 0.0000045 0.000000 0.000021 0.000000 0.001323 -0.002373 0.000000 0.000000 0.000000
	lupeol T3 A3 T3 β amyrin B12 Epi αamyrin β amyrin		0.047 0.01001 0.00968 0.0001339 0.000000 0.000000 -0.000039	0.00000 0.02364 0.000000 0.000000 0.000118	0.00000 0.000000	0.00000 0.000000
	Variable Epi βamyrina Epi lupeol A2 Epi lupeol αamyrin/β amyrona P1 β amyrin αamyrin/β amyrona	PC8 0.010 0.09710 0.01307 -0.0000834 0.000000 0.000000 0.000000	PC9 0.0020377 PC10 0.16474 0.05573 PC11 0.000000 0.000251 0.000000 0.000000	PC12 0.04070 0.000584 -0.000971 PC13 PC14 0.00817 Epi
	A4 lupenone lupeol A1 lupeol α amyrone P2 αamyrin/β amyrona α amyrone	0.113 -0.048 0.001 0.01927 0.0600 0.0000022 0.0000956 0.317 0.00870 0.03533 0.208 -0.152 0.02352 0.273 0.0005593 0.000000 0.000000 0.000000 0.000000 0.000000 0.000000 0.000000 0.358 0.01635 -0.000002 -0.000009 B12 P1 P2 B14 B15 E1 D1 T3 lupeol
	A3 Epi Epi βamyrina αamyrin B11 Epi βamyrina T1 B14 α amyrone T1 A4		0.289 0.153 0.00899 0.338 0.0000 0.0000134 0.0000551 0.090 -0.087 0.00975 0.00000 0.0000515 0.343 0.00337 0.304 -0.509 0.01753 0.0000478 0.000000 0.000000 0.000000 -0.000011 0.000000 -0.000062 0.000104 0.000000 0.000000
	A2 β amyrin lupenone B12 lupenone B15 T1 lupeol A3		0.367 -0.088 0.116 0.03922 0.0000 -0.0000313 0.0001601 0.148 -0.291 -0.465 -0.176 -0.242 0.07266 0.01457 0.00649 0.01565 0.0000125 0.0000162 0.0000883 0.000000 0.000000 0.000000 0.000000 0.000000 0.000000 0.000000
	A1 αamyrin/β amyrona 0.226 0.188 Epi αamyrin 0.01169 0.222 P1 0.000000 Epi αamyrin -0.0000037 0.0000604 0.073 -0.086 -0.125 -0.316 0.01895 0.00000 0.00176 0.0000500 0.0000166 E1 0.000000 0.000000 P2 0.000000 Epi βamyrina A2	0.416 0.00894 0.0000175
	B1 α amyrone β amyrin P2 β amyrin D1 B14 lupenone A1		0.014 -0.458 -0.234 0.128 0.04328 0.07863 0.00561 0.062 0.07824 -0.0000647 0.0001409 0.0000632 0.456 0.00390 0.047 0.0000183 0.000000 0.000000 0.000000 0.000257	0.042 0.01978 0.0000788
	B2 T4 αamyrin/β amyrona B14 αamyrin/β amyrona -0.0000486 -0.566 0.534 0.042 0.04688 0.00000 0.0001880 -0.0000006 0.021 0.258 0.026 -0.138 -0.023 0.08861 0.00000 0.00244 0.05198 0.0000030 0.0000755 Covariance matrix for Group bipinnata T3 0.000000 0.000000 B15 0.000000 0.000257 0.000257 Epi αamyrin B11
	B3 α amyrone B15 α amyrone Epi lupeol E1 β amyrin B12		-0.023 0.01139 0.107 0.000000 -0.0000023 0.0000246 0.150 -0.017 -0.128 0.00761 0.00043 0.00294 0.068 -0.267 0.02862 0.0000232 0.0000101 0.0000078 0.000000 0.000000 0.000000 0.000000 0.000000 0.000000 0.036971
	B4 T1 E1 T1 lupeol D1 αamyrin/β amyrona P1	-0.102 -0.233 0.00259 0.000000 -0.0000108 -0.0000091 0.143 0.00000 0.00000 0.256 -0.203 0.00000 0.540 -0.060 0.00000 0.0000005 0.0000032 0.0000045 0.000000 0.000000 0.000000 0.000266 0.000266 0.000000 0.000283 0.000000 0.000000 0.000000
	B5 D1 β amyrin Epi βamyrina T3 α amyrone P2		-0.045 0.00000 0.101 0.0002318 A4 0.000000 0.000000 0.074 -0.062 -0.182 A3 A2 0.000000 0.000291 0.000291 0.000000 0.011 -0.157 A1 B11 0.000307 0.000000 0.000000 0.000000 0.000000
	B6 T3 αamyrin/β amyrona -0.0000008 -0.349 -0.134 -0.269 -0.209 -0.292 0.02289 0.0002123 A4 0.000093 lupenone 0.000000 0.000000 Epi lupeol 0.000000 0.000194 0.000194 0.000000 0.181 0.000139 0.090 T1 0.000000 0.000000 B14 0.000000 0.000000
	B7 Variable Epi lupeol α amyrone A3 Epi αamyrin lupeol T1 B15		0.040 -0.158 stenophylla 0.07423 0.0000229 0.0000022 0.582 0.000101 0.000588 0.000000 0.000000 0.000000 0.000025 0.000000 0.000000	0.064 0.0000199 0.159 -0.159 0.000025 0.000000 -0.000029 0.043
	B8 A4 lupeol T1 A2 β amyrin Epi βamyrina E1		-0.127 0.08870 0.149 0.00584 0.0000057 0.0000001 -0.0000010 0.168 -0.316 -0.111 0.0000120 0.396 0.000027 0.000197 0.000131 0.000000 0.000000 0.000000 0.000000 0.000000 0.000000 0.000000 0.068 0.000000 0.000000
	T3 A3 Epi βamyrina A1 αamyrin/β amyrona lupenone D1	-0.440 -0.278 0.02406 0.005076 0.000010 -0.000412 -0.000254 0.374 -0.103 -0.000 -0.208 -0.251 0.006025 0.000000 0.000000 0.000000 0.000200 0.000200 0.000000 0.000212 0.001335 0.000000
	Epi lupeol A2 lupenone B11 α amyrone Epi αamyrin Covariance matrix for Group laxiflora -0.011 0.099 -0.164 -0.181 -0.131 -0.099 -0.040 0.02495 0.012565 Epi 0.000000 0.000000 0.000000 0.000000 0.000000 0.000000 0.000000 0.000000 0.000000 0.000000 0.000000 0.000000 T3 0.000000 0.000000
	lupeol A1 Epi B12 T1 β amyrin αamyrin Epi lupeol		0.233 0.20237 0.203 -0.045 0.004465 B12 P1 P2 B14 B15 E1 D1 T3 lupeol lupeol 0.569 -0.117 0.051 0.012 0.000000 0.000000 0.000000 0.000000 0.000000 0.000000 0.000000 0.000000 -0.000020 -0.000020 0.000000 -0.000035 0.000829 0.000000
	Epi βamyrina B11 β amyrin A4 P1 P2 αamyrin/β amyrona lupeol	0.013 0.00000 0.231 0.02010 0.000000 0.000000 0.148 -0.424 0.000000 0.000000 0.000000 0.000000 -0.001044 0.000000	0.344 0.000000 0.000000 0.194 0.366 0.000000 0.000000
	lupenone B12 αamyrin/β amyrona A3 P2 B14 α amyrone Epi βamyrina	-0.058 0.00000 0.005 -0.141 -0.083 0.01664 -0.000574 -0.001066 -0.000472 -0.002252 0.000000 0.009 0.041 -0.092 0.000000 -0.000003 -0.000003 0.000000 -0.000003 A4 A3 A2 A1 B11 -0.000109 0.000000
	Epi P1 α amyrone αamyrin A2 B14 B15 T1 A4 lupenone		-0.015 0.00000 0.028 0.005649 0.000019 0.000006 -0.000036 0.069 -0.004 0.000000 0.000000 0.000000 0.001279 -0.000012 0.000000	0.159 -0.163 -0.191 0.000035 0.000000 0.000000 0.000000
	P2 T1 A1 B15 E1 lupeol A3 Epi	αamyrin		0.10061 0.007750 0.000000 0.000620 0.001413 -0.000084	0.000000 0.002082 0.000000	0.000000	0.000000 0.000000
							241 242 243 245 247

Concentration of Standard Molecules Calculated from peak areas and correspondent equations from calibration

		Annex 3 Annex 3 Annex 4 Annex 4 Annex 4 Annex 4 Annex 4 Annex 4
	Epi βamyrina E1 B. grandifolia markers 0.000058 -0.000299 0.000000 0.000000 0.000000 Mass Spectra of Identified Triterpenoids in Mexican Copals 0.000000 0.000000 CH 3 CH 3 H 3 C CH 3 H 3 C CH 3 H 3 C B. grandifolia markers B. simaruba markers B. simaruba markers
	lupenone Epi αamyrin β amyrin αamyrin/β amyrona α amyrone T1 lupeol Epi βamyrina lupenone Epi αamyrin β amyrin αamyrin/β amyrona -0.000041 0.000092 -0.000078 0.000000 0.000012 -0.000076 0.000000 -0.000019 -0.000230 0.000000 0.000429 0.000035 0.000000 0.000059 -0.000073 0.000000 0.000000 0.000000 0.000000 0.000302 0.000221 0.000237 0.000100 0.000078 0.000059 0.000085 0.000061 0.000031 0.000353 0.000118 0.000109 0.000050 0.000104 -0.000002 -0.000200 0.000000 0.000000 0.000000 0.000000 0.000000 0.000000 0.000000 0.000000 0.000000 0.000000 0.000000 0.000000 0.000025 0.000102 0.000700 α amyrone 0.000040 0.000061 0.000024 0.000012 -0.000009 T1 0.000000 0.000000 0.000000 0.000000 0.000000 T1 0.000000 B12 P1 P2 B14 B15 E1 D1 T3 lupeol Epi A4 A3 A2 A1 B11 B12 0.000000 P1 0.000000 0.000226 P2 0.000000 0.000236 B14 0.000000 0.000134 B15 0.000000 0.000000 E1 0.000000 0.000000 D1 0.000000 0.000000 T3 0.000000 0.000000 Epi lupeol 0.000000 -0.000016 lupeol 0.000000 -0.000071 Epi βamyrina 0.000000 -0.000070 lupenone 0.000000 0.000045 Epi αamyrin 0.000000 -0.000012 β amyrin 0.000000 -0.000043 αamyrin/β amyrona 0.000000 0.000326 α amyrone 0.000000 0.000010 T1 0.000000 0.000000 P2 B14 B15 Covariance matrix for Group stenophylla A4 A3 A2 A1 B11 A4 0.000843 A3 -0.000102 0.000094 A2 0.000000 0.000074 0.000171 A1 0.000392 0.000144 0.000156 0.003594 B11 0.000000 0.000000 0.000000 0.000000 0.000000 B12 0.000000 0.000000 0.000000 0.000000 0.000000 P1 0.000000 0.000000 0.000000 0.000000 0.000000 P2 -0.001543 0.000572 0.000288 -0.000428 0.000000 B14 0.000000 0.000000 0.000000 0.000000 0.000000 B15 0.000000 0.000000 0.000000 0.000000 0.000000 E1 0.000000 0.000000 0.000000 0.000000 0.000000 D1 0.000000 0.000000 0.000000 0.000000 0.000000 T3 0.000297 0.000111 0.000041 0.000681 0.000000 Epi lupeol 0.001137 -0.000543 -0.000109 -0.001803 0.000000 lupeol 0.000092 -0.000029 -0.000033 0.000067 0.000000 Epi βamyrina -0.000005 0.000024 -0.000006 -0.000057 0.000000 lupenone -0.000216 -0.000057 -0.000068 -0.000388 0.000000 Epi αamyrin -0.000042 -0.000005 -0.000027 -0.000139 0.000000 β amyrin -0.000127 -0.000110 -0.000182 -0.000775 0.000000 αamyrin/β amyrona -0.000212 -0.000067 -0.000028 -0.000245 0.000000 α amyrone 0.000097 -0.000034 -0.000040 -0.000075 0.000000 T1 -0.000076 0.000015 0.000014 -0.000136 0.000000 P2 0.006121 B14 0.000000 0.000000 B15 0.000000 0.000000 0.000000 E1 0.000000 0.000000 0.000000 0.000000 D1 0.000000 0.000000 0.000000 0.000000 0.000000 T3 -0.000001 0.000000 0.000000 0.000000 0.000000 Epi lupeol -0.004780 0.000000 0.000000 0.000000 0.000000 lupeol -0.000366 0.000000 0.000000 0.000000 0.000000 Epi βamyrina 0.000081 0.000000 0.000000 0.000000 0.000000 lupenone 0.000055 0.000000 0.000000 0.000000 0.000000 Epi αamyrin -0.000035 0.000000 0.000000 0.000000 0.000000 β amyrin 0.000008 0.000000 0.000000 0.000000 0.000000 αamyrin/β amyrona -0.000332 0.000000 0.000000 0.000000 0.000000 α amyrone -0.000165 0.000000 0.000000 0.000000 0.000000 T1 0.000088 0.000000 0.000000 0.000000 0.000000 lupeol 0.000034 D1 0.000000 0.000000 T3 0.000000 0.000000 Epi lupeol 0.000000 0.000000 lupeol 0.000000 0.000000 Epi βamyrina 0.000000 0.000000 lupenone 0.000000 0.000000 Epi αamyrin 0.000000 0.000000 β amyrin 0.000000 0.000000 αamyrin/β amyrona 0.000000 0.000000 α amyrone 0.000000 0.000000 T1 0.000000 0.000000 P2 B14 B15 E1 D1 T3 0.000524 Epi lupeol -0.000483 0.005510 lupeol 0.000032 0.000207 Epi βamyrina 0.000067 -0.000094 lupenone -0.000258 0.000149 Epi αamyrin -0.000018 0.000046 β amyrin -0.000307 0.000273 αamyrin/β amyrona -0.000243 0.000485 α amyrone -0.000011 0.000145 T1 -0.000054 -0.000045 lupeol Epi βamyrina lupenone Epi αamyrin β amyrin αamyrin/β amyrona 0.000277 α amyrone -0.000039 0.000032 T1 0.000000 -0.000005 T1 Summary of Classified Observations Sample Lupeol Lupenone ß-amyrin α -amyrone 11a1 .03269 .20992 .21902 .02859 11b2 .02242 .28544 .203043 .014324 11b3 0.29493 1.8014 1.917769 0.14504 11c1 .07075 .32878 .33903 .02074 11c2 0.027865 0.320973 0.332013 0.00996 11c3 .199096 1.21896 1.177103 .0566 11d1 .2442 .09025 .109128 .016385 11d2 .339180 01/01/60 1.743341 .31403 11d3 .0403 .299999 .148078 .04568 11e1 .067099 .697966 .45539 .153278 27a2 0.57309 0.077511 0.07435 0.00197 27a3 0.02679 0.02466 0.00565 0.00207 27a4 0.07678 0.08585 0.00758 nd 27a1Q 0.61844 0.07561 0.10660 0.00134 47a1 0.00268 0.00961 0.00939 nd 47a2 1.08655 0.2633 0.32767 0.11788 47b2 0.01092 0.01553 0.001152 0.0098 47b3 0.047712 0.01378 0.00817 47b4 0.00993 0.16302 0.4594 47b5 nd 0.00316 nd 47b6 0.13038 0.58163 0.05887 47d1 0.00023 0.0098 0.02506 0.003359 47c1 0.0153 0.005961 0.01463 0.001853 47ee1 0.02586 0.00147 nd 0.014554 47f1 0.22704 nd nd 0.0266 75% 75% 75% 50% 50% 50% 100% 392.8 1.089e+6 Spectrum 2A 32.771 min, Scans: 2060-2062, 40:650, Ion: 135 us, RIC: 9.037e+6, BC BP: 392.8 (1.089e+6=100%), 70a1q f1 1.2mg 1ml 2µl 345736 100% 203.0 34.885 min, Scan: 2194, 40:650, Ion: 182 us, RIC: 6.451e+6, BC BP: 203.0 (345736=100%), 70a1q f1 1.2mg 1ml 2µl triterpensspl 87933 100% 189.0 Spectrum 1A Spectrum 1A 38.321 min, Scan: 2409, 40:650, Ion: 865 us, RIC: 1.249e+6, BC BP: 189.0 (87933=100%), 70a1q f1 1.2mg 1ml 2µl triterpensspl tr = 32.77 min 0.0437 tr = 34.88 min tr = 38.32 min GS1 nd 100% 218 75% 75% B. grandifolia-simaruba markers S1 S3 0.06 100% 218 44.893 min. Scans: 2826-2828 Chan: all Ion: NA RIC: 1668023 BC 75% B. simaruba markers 0.0052 α α α α-Amyrenone C 30 H 48 O 424 g.mol -1 Commercial compoundt: BCP Gradient 1 tr = 43.15min Gradient 2 tr = 35.87min CH 3 H CH 3 CH 3 CH 3 H CH 3 CH 3 H 3 C H 3 C O Spect 1 Spect 1 44.218 min. Scans: 2810-2812 Chan: all Ion: NA RIC: 276999 BC α α α α-Amyrine TMS C 33 H 58 OSi 498 g.mol -1 Commercial compound: Extrasynthèse Gradient 1 tr = 43.58 min Gradient 2 tr = 36.24 min (H 3 C) 3 SiO CH 3 H H 3 C CH 3 G3 GS2 S2 S4 CH 3 CH 3 tr = 38.12 min tr = 37.69 min tr = 35.64 min tr = 38.32min CH 3 CH 3 CH 3 H CH 3 H 3 C β β β β-Amyrine TMS C 33 H 58 OSi 498 g.mol -1 Commercial compound: Extrasynthèse Gradient 1 tr = 42.81 min Gradient 2 tr = 35.24min (H 3 C) 3 SiO CH 3 H CH 3 CH 3 H CH 3 H 3 C 100% 203 Spect 1 36.846 min. Scans: 2318-2320 Chan: all Ion: NA RIC: 1083669 BC 3-epi-β β β β-amyrine C 33 H 58 OSi 498 g.mol -1 Isolated product Gradient 1 tr = 40.50min Gradient 2 tr = 32.53min 100% 203 Spect 1 41.396 min. Scans: 2635-2637 Chan: all Ion: NA RIC: 4684733 BC (H 3 C) 3 SiO CH 3 H CH 3 CH 3 H CH 3 H 3 C (H 3 C) 3 SiO 3-epi-lupeol TMS C 33 H 58 OSi 498 g.mol -1 Isolated product Gradient 1 tr = 41.12 min Gradient 2 tr = 33.22 min 100% 100% 100 200 300 400 500 100 200 300 400 500 m/z 600 m/z 600 100 200 300 400 500 600 m/z 0% 100 200 300 400 500 m/z 600 203 0% 189 Spect 1 41.868 min. Scans: 2662-2664 Chan: all Ion: NA RIC: 7967257 BC β β β β-amyrone C 30 H 48 O 424 g.mol -1 Commercial product : BCP instruments Gradient 1 tr = 42.48 min Gradient 2 tr = 34.76 min CH 3 H 3 C Spect 2 43.829 min. Scans: 2756-2758 Chan: all Ion: NA RIC: 38710 BC 25% 50% 75% 41.0 11694 73.0 27797 79.0 9041 95.0 16705 120.0 5074 159.0 7661 160.9 17338 24778 201.8 4776 202.8 15982 296.8 5135 421.7 14616 422.6 5654 497.8 4954 0% 25% 50% 65.0 9722 25751 106.0 6651 122.9 145.8 4517 189.8 9374 244.8 10962 313.7 6321 380.7 5593 424.7 9778 25% 73.0 106965 119.1 54327 0% 73799 81758 173.0 241.0 108479 393.7 170857 482.7 77.1 9257 123.0 8585 173.0 12074 13305 8462 10104 296.8 497.8 228.8 25% 18477 203.8 21553 312.7 25619 50% 23579 25129 107.0 27349 69.0 175.0 79.0 39375 135.0 33681 32400 50% 39912 423.7 73.1 190.0 75% 91.0 75% 53643 47021 190.0 41.0 52258 75% H CH 3 CH 3 CH 3 94.9 55700 O 100% 189.0 204.7 589472 100% 392.7 45261 Spectrum 1A Spectrum 1A Spectrum 1A 38.120 min, Scan: 2361, 40:650, Ion: 889 us, RIC: 841853, BC 100% 108.9 71355 Spectrum 1A 35.642 min, Scan: 2241, 40:650, Ion: 248 us, RIC: 4.793e+6, BC BP: 392.7 (589472=100%), 70a1q f1 1.2mg 1ml 2µl triterpensspl 85780 100% 189.0 BP: 189.0 (45261=100%), 34b1q triterpens.sms 37.697 min, Scan: 2335, 40:650, Ion: 539 us, RIC: 1.675e+6, BC BP: 108.9 (71355=100%), 34b1q triterpens.sms 38.321 min, Scans: 2408-2410, 40:650, Ion: 886 us, RIC: 1.199e+6, BC BP: 189.0 (85780=100%), 70a1q f1 1.2mg 1ml 2µl triterpensspl
	E1 D1 T3 Epi lupeol lupeol Epi βamyrina lupenone Epi αamyrin β amyrin αamyrin/β amyrona 0.000000 -0.000001 0.000000 0.000000 0.000060 0.000000 0.000133 0.000000 0.000094 0.000000 0.000049 0.000000 0.000038 0.000000 0.000166 α amyrone 0.000000 0.000018 T1 0.000000 0.000000 lupeol Epi βamyrina lupenone Epi αamyrin β amyrin αamyrin/β amyrona 0.000652 α amyrone 0.000072 0.000023 T1 0.000000 0.000000 T1 Epi βamyrina 0.000006 0.000026 lupenone -0.000018 -0.000026 47a1Q 0.14343 0.002012 0.009985 189 121 50% 95 75% 0.000158 Epi αamyrin 0.000010 0.000014 0.000019 0.000020 β amyrin -0.000006 -0.000015 0.000195 0.000036 0.000404 0.007235 αamyrin/β amyrona -0.000008 -0.000039 0.000154 47b1Q 0.23616 0.087744 0.02957 0.02192 63a1 0.00089 0.022623 0.01754 0.041073 50% 50% 25% 394 75% 190.0 75% 53715 217.8 0.000015 0.000077 α amyrone 0.000014 -0.000002 0.000000 0.000001 0.000065 T1 0.000003 0.000016 0.000022 0.000023 0.000029 T1 0.000060 Epi B12 P1 P2 B14 B15 E1 D1 T3 lupeol A4 A3 A2 A1 B11 B12 0.000000 P1 0.000000 0.000000 P2 0.000000 0.000000 B14 0.000000 B15 0.000000 0.000000 100 200 300 400 0.000000 63a2 0.00452 0.045533 0.024 0.077333 63a3 0.08219 0.93544 0.12598 25% 73 206263 73.1 25% 50% 41407 0.02622 63a4 0.05625 0.126608 0.04404 0.102615 63a1Q 0.183031 0.092325 0.08407 0.23138 63b1Q 0.0022 0.012729 0.01163 0.084771 72a1 0.09719 0.26849 0.09716 0.104671 72a2 0.05368 0.270574 0.26394 0.03376 72b1 0.10936 0.315295 0.42913 72b2 0.02281 0.155033 nd 0.029475 72c1 0.040643 0.173141 0.22273 0.047922 50 100 150 200 250 300 350 0% 313 341 100 200 300 400 500 600 100 200 300 400 500 600 0% 257 337 365 393 100 200 300 400 0% 100 200 300 400 500 600 0% 0.102815 25% 55 122 147 258 281 25% 73 122 147 0% 25% 73 147 257 337 365 393 100 200 300 400 0% 147 257 366 394 100 200 300 400 0% 257 297 339 369 100 200 300 0% 41 95 299 323 25% 50% 69.0 189505 133.1 91211 189.0 259268 255.0 90220 393.7 320161 482.4 81394 25% 50% 41.0 95.1 141952 147.0 190.9 129978 408.8 147401 175.0 69.0 107.0 27521 26219 23633 103989 43.0 40818 94.2 0% 30847 100994 148.0 38788 204.8 64070 256.8 74487 310.8 52675 409.7 52844 25% 55.0 7842 94.1 7270 149.0 10347 228.8 188.0 10873 14135 497.8 296.8 8561 9530	m/z 500 m/z 499 500 m/z 425 499 500 m/z 499 498 500 m/z 499 m/z 410 400 m/z m/z 455 m/z 456 400
		250 251 264 265 275 276 277 278

Fig 120 B. simaruba and B. grandifolia mass spectra of molecular markers B. penicillata markers

  Scan: 2492, 40:650, Ion: 458 us, RIC: 2.223e+6, BC BP: 423.3 (155531=100%), 63a4 2.3mg 1ml 1µl triterpenes 14-02-58

								Annex 4 Annex 4 Annex 4
	B. penicillata markers B. penicillata markers			P6
	tr = 39.51 min			P2 P4
	tr = 37.93 min tr = 38.65 min Spectrum 1A
	0% 25% 50% 75% 100% 0% 25% 50% 75% 100% 0% 25% 50% 75% 100%	100 91.3 73.2 21974 9371 117.4 26474 132.4 40946 133.4 12417 Spectrum 1A 41.1 6868 BP: 202.5 (72412=100%), 63a4 2.3mg 1ml 1µl triterpenes 14-02-58 200 300 189.6 6313 201.7 19474 202.5 72412 241.9 4311 100 200 300 45.1 5232 73.2 34200 75.2 13975 91.3 6700 129.3 10101 134.5 3044 161.6 18551 173.5 9126 203.8 21179 216.8 42385 217.7 10869 229.8 3072 269.8 2215 Spectrum 1A 38.655 min, Scans: 2438-2440, 40:650, Ion: 1016 us, RIC: 703710, BC 400 500 600 m/z 409.3 26170 410.4 9157 37.938 min, Scans: 2394-2396, 40:650, Ion: 949 us, RIC: 852581, BC 400 500 600 m/z 392.3 5963 497.8 30495 498.8 13311 499.9 4824 BP: 216.8 (42385=100%), 63a4 2.3mg 1ml 1µl triterpenes 100 200 300 400 500 600 m/z 55.1 24653 73.1 73098 133.4 40137 149.4 11461 203.7 84868 205.6 13568 216.7 44175 353.1 42342 354.2 13985 409.4 14107 423.3 155531 424.3 51022 497.8 8181 39.517 min, P7
	tr = 40.00 min
	P3 P5 P1 38.248 min, Scans: 2413-2415, 40:650, Ion: 312 us, RIC: 3.496e+6, BC 39.191 min, Scans: 2471-2473, 40:650, Ion: 904 us, RIC: 920634, BC BP: 216.8 (57666=100%), 63a4 2.3mg 1ml 1µl triterpenes 14-02-58 Spectrum 1A Spectrum 1A BP: 423.2 (279921=100%), 63a4 2.3mg 1ml 1µl triterpenes Spectrum 1A 158433 203.6 tr = 37.13 min 100% tr = 38.25 min Spectrum 1A tr = 39.19 min 39.993 min, Scans: 2520-2522, 40:650, Ion: 405 us, RIC: 2.587e+6, BC BP: 203.6 (158433=100%), 63a4 2.3mg 1ml 1µl triterpenes
	100% 100% 100% 75%	202.7 BP: 202.7 (125750=100%), 63a4 2.3mg 1ml 1µl triterpenes 216.8 57666 125750	37.132 min, Scans: 2344-2346, 40:650, Ion: 1198 us, RIC: 625525, BC 423.2 279921 467.5 46049 409.3 109130
	75% 75% 75%	73.2 88251	202.7 88358
	25% 50% 0% 25% 50% 50% 50% 0% 0% 25% 25%	73.2 28399 55.1 33022 73.2 73.2 118247 41.0 5472 9182 100 119.3 132.4 19262 161.6 26391 133.5 48009 190.4 203.6 22387 203.8 216.8 143132 31133 52819 163.5 23069 215.7 28949 157.4 4423 229.7 5416 269.8 200 3663 56150 175.7 81701 22883 81.3 119.3 12805 187.7 14349 69.0 26888 143.5 18442 14670 215.7	326.1 17749 20839 300 306.8	483.7 7825 500 5411 497.9 468.5 452.6 3821 86000 410.4 49539 400 9717 424.3 407.4 15775 513.1 20487 392.1 31297	600	m/z
	0%
		100 100 100	200 200 200	300 300 300		400 400 400	500 500 500	600 600 600	m/z m/z m/z
	B. penicillata markers
								279 280 281

Fig 121 B. penicillata mass spectra of molecular markers B. copallifera markers

  Scan: 2121, 40:650, Ion: 1620 us, RIC: 419842, BC BP: 203.3 (36303=100%), b copallifera tritpns 2µl14-46-27 25-11-2011 Scans: 2422-2424, 40:650, Ion: 1363 us, RIC: 653666, BC BP: 203.3 (34806=100%), b copallifera tritpns 2µl 14-46-27

									Annex 4 Annex 4 Annex 4
	B. copallifera markers				C1 C5
	tr = 33.72 min 0% 25% 50% 75% 100% 41.1 9258 43.0 2221 tr = 38.50 min Spectrum 1A tr = 34.64 min 0% 25% 50% 75% 100% 55.1 13333 65.1 3291 Spectrum 1A BP: 218.1 (49886=100%), b copallifera tritpns 2µl 14-46-27 100 200 300 95.2 7175 133.2 5863 189.3 12797 203.3 36303 205.2 5970 218.2 22626 257.3 3540 311.3 2252 C3 106.2 4935 119.2 13830 161.2 13191 174.2 4078 203.3 25901 218.1 49886 219.2 9220 257.2 7422 34.645 min, Scans: 2177-2179, 40:650, Ion: 1015 us, RIC: 736528, BC 400 500 600 m/z 406.4 3679 409.3 9595 393.2 13918 394.5 4210 100 200 300 400 500 600 m/z 0% 25% 50% 75% 100% 41.1 6745 73.0 23683 91.3 7369 133.1 9501 159.2 3863 190.3 13094 202.3 18830 203.3 34806 204.2 7437 306.3 6161 391.6 6103 408.5 33421 409.4 17954 511.8 8092 Spectrum 1A 33.722 min, C2 38.502 min,
	tr = 34.12 min	100	200	300		400	500	600	m/z
		Spectrum 1A
	100% tr = 35.03 min BP: 203.3 (79609=100%), b copallifera tritpns 2µl 14-46-27 25-11-2011 C4 203.3 79609	34.123 min, Scan: 2146, 40:650, Ion: 730 us, RIC: 1.118e+6, BC
		Spectrum 1A
	100% 75%	218.0 52593 130110 218.0 BP: 218.0 (130110=100%), b copallifera tritpns 2µl 14-46-27	35.030 min, Scans: 2201-2203, 40:650, Ion: 737 us, RIC: 1.154e+6, BC
	50% 75%			204.2 35791
	25% 50% 0% 25%	41.1 15172 55.1 73.0 37007 10629	100 105.2 20545 122.0 7608 160.3 163.2 189.3 200 18454 202.3 68241 10006 4256 190.3 35205 119.2 19814 204.2 8728	300		400 408.4 13419 393.2 7416	500 498.8 7498	600	m/z
	0%
			100	200	300		400	500	600	m/z
									282 283 284

For this research we have chosen to study both the totality of the resins for its convenience for molecular profiling when applying the statistical methods of PCA and LDA, and the tritrepenic fraction because of the resistance of these compounds to degradation, and its possible utility as molecular markers.

The first difficulty for the study of resins by HPLC arisen, when studying samples from certified botanical origin, because of the need of different amounts of sample depending on their terpenoid content of the studied species: from 2 mg up to 30 mg, refer to table 10.

Type of sample

Mass of sample used for HPLC analysis As can be seen on fitting matrix this model seems to be more robust than the one constructed with FTIR data, affording a 100% of correct recognition. Complete statistical results can be consulted in annex 3.

The confusion matrix (table 19) afforded 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 affords as a consequence that when dealing with classification from a sample of B.

stenophylla 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 the graphic 69 a greatest dispersion of the B. stenophylla data in the hyperspace can be noticed, this fact may be correlated to a greater possibility of misclassification.

HPLC coupled to PCA analysis for archeological resins

When HPLC data for archaeological samples was introduced into the model, a good discrimination was achieved between the archaeological sample from Chichén Itzá and the archaeological samples from Templo Mayor, this comes as a logical consequence of their great molecular difference, corroborated by HPLC chromatograms (figure 72).

ANALYTICAL STUDY OF THE RESINS AND DISCUSSION ANALYTICAL STUDY OF THE RESINS AND DISCUSSION

PAOLA LUCERO

113 Finally concerning botanical origin determination we hypothesized that the most probable origin for the 84 sample from Templo Mayor is likely to be B. stenophylla, as its position in the PCA graphic is close to the region of the hyperspace, where this species is distributed (figure 73).

First Component

Second Component

HPLC-PCA data with archeological samples

Concerning the other samples of Templo Mayor it was not possible, to assess with certainty their botanical origin, given the botanical closeness of the species, and the many molecular changes that take place during ageing processes, giving place to to the disparition of some compounds sometimes (A2, A3, B8, epi-β-amyrin, α-amyrone, β-amyrone) or always (B1, B2, B3, B5) and the apparition of others (C1 to C4).

ANALYTICAL STUDY OF THE RESINS AND DISCUSSION ANALYTICAL STUDY OF THE RESINS AND DISCUSSION

PAOLA LUCERO

114 In order to achieve a better botanical discrimination in aged samples further studies need to be conducted into degradation processes, comprising an identification of both volatile molecules and molecules arisen in aged samples by means of R.M.N.

First Component

Second Component

HPLC-PCA data with archeological samples

In a second step the establishment of correlations between products of degradation and stages of ageing could be established. This type of work was beyond the scope of the present work, because of time constraints.

ANALYTICAL STUDY OF THE RESINS AND DISCUSSION ANALYTICAL STUDY OF THE RESINS AND DISCUSSION

PAOLA LUCERO

123 described in the materials and methods section, compounds of a same family are always eluted according to the following order: 3-α-alcohol, 3-ketone and 3-β-alcohol. A depth comment on fragmentation patterns of triterpenes can be found in Mathe, 2004.

Results of the study on botanical certified samples

The main objective of this type of work was to establish a molecular profile, to distinguish chemically resins from different botanical origin, and to identify as many compounds as possible into their chromatograms.

As in the HPLC study, for the study of certified samples, different amounts of sample depending on the studied species were needed (as much as possible the amount of sample was tried to be kept at 2 mg). The amount used for each different botanical origin of the resins is given in table 22. Some general observations about composition of resins will be address on this section, but the main analysis of triterpenic fraction will be done on next section of this chapter, using protocol 2 chromatograms.It is worthy to comment some correlations and differences between HPLC and GC chromatograms, as striking difference can be remarked between GC chromatograms and HPLC ones. Somewhere between B. excelsa that is rather poor in triterpenes and B. penicillata that is really rich on them.

Studied species

B. bipinnata B. stenophylla

Although our study was not focused on the characterization of the compounds on the volatile fraction of these resins, some observations can be made. 78).

Concerning B. penicillata its triterpenoid fraction seems to be much richer than we initially suppose after HPLC analysis.

The same happened with B. simaruba, its richness of triterpenoid content of could be the cause of the difficulties in the resolution of some peaks in HPLC chromatograms (figure 79).

B. simaruba and B. grandifolia species exhibit a moderate variety of molecules in the volatile fraction of the resin. Finally and although B. copallifera was not part of the original study we would like to address a commentary on its composition: it seems to have very rich terpenoid tenure in qualitative terms (figure 80). Although their concentration is limited, volatile fraction (sesqui-and monoterpenoids) seems to include a number of molecules as well.

B. penicillata B. simaruba

B. grandifolia B. copallifera

An in depth commentary on the composition of triterpenic fraction will be addressed in section 5.5 of this chapter.

Annexes

Annex 1

186

B. bipinnata, B. excelsa and B. grandifolia

ABSTRACT

In the present work, the molecular composition of series of resinous Aztec and Maya archaeological samples were investigated to determinate their nature and botanical origin.

Thus an analytical strategy was specifically designed. This analytical strategy included the analysis of botanically certified resins, freshly collected. It included as well the analysis of commercial samples bought in Mexican traditional markets.

The study of all the samples included microscopical techniques, Fourier Transformed Infrared Spectrometry (FTIR) , High Performance Liquid Chromatography coupled to Ultraviolet-visible spectrometry (HPLC-UV/Vis) and Gas Chromatography coupled to Mass Spectrometry (GC-MS).

The molecular study of these samples, in particular of their triterpenic composition allowed to: ◙ ◙ Establish the molecular profile of resins from certified botanical origin.

◙ ◙ Identify some of the triterpenic compounds present in samples. ◙ ◙ Asses that botanical origin of a sample can be found regardless the age of the resin.