Chemical Composition and Biological Activities of Fragrant Mexican Copal (Bursera spp.)

Copal is the Spanish word used to describe aromatic resins from several genera of plants. Mexican copal derives from several Bursera spp., Protium copal, some Pinus spp. (e.g., P. pseudostrobus) and a few Fabaceae spp. It has been used for centuries as incense for religious ceremonies, as a food preservative, and as a treatment for several illnesses. The aim of this review is to analyze the chemical composition and biological activity of commercial Mexican Bursera copal.


Introduction
The term "resin" is often used to describe fragrant plant saps or exudates distinguished from other plant exudates such as gums, mucilages, oils, waxes, and latex. Plant resin is defined primarily as "a lipid-soluble mixture of volatile and non-volatile terpenoid, and/or phenolic secondary compounds that are (a) usually secreted in specialized structures located either internally or on the surface of the plant and (b) of potential significance in ecological interactions" [1,2]. Resins usually consist of a volatile fragrant fraction, usually called essential oil, and a non-volatile fraction, usually consisting of long-chain terpenoids. When fresh resins are translucent liquids but with time and the loss of the essential oil fraction, they turn into brown, yellow, or white solids that, by polymerization and oxidation, fossilize as amber [3].
Resins have been used since ancient times as constituents of varnishes, cosmetics, adhesives, and as incense in ritual ceremonies in temples and churches. Resins from three important genera of the Burseraceae-Boswellia, Commiphora, and Bursera-have been, and still are, used in perfumery and particularly as incense. Boswellia resin is called frankincense, Commiphora resin is commonly known as myrrh and Bursera resin is often referred to as copal.
The word copal derives from copalli, the Náhuatl (Atzec) term for incense. The Maya, in turn, used the term pom [4,5] for the incense derived from Protium, Bursera, and Pinus, depending on which resin-producing trees were most abundant in the areas where they lived. Later, the Spanish exported the term copal to Europe [1]. Nowadays, outside Mexico, the term is used for resins of the Fabaceae family and, generically, resins from Burseraceae are sometimes called elemi [6]. In Mexico and Guatemala copal derives mostly from Bursera, from a species of Protium (Protium copal) and a pine species (Pinus pseudostrobus) [3]. Bursera's distribution encompasses tropical regions from southern United States (southern Arizona, California, and Florida) to Peru. In Mexico, the genus is highly diverse and abundant along the Pacific slopes [1]. Protium copal and Pinus pseudostrobus are found in Mexico and Central America.
In marketplaces of Central Mexico it is possible to find a variety of copal types to satisfy many tastes (and budgets, as prices vary according to the quality): copal blanco, copal oro, copal negro, Table 1. Comparison of a fresh sample of Bursera cuneata copal (C), a five-year-old sample (D), and samples given two different artificial treatments (sample C.1, prepared by dissolving resin in turpentine and sample C.2, obtained heating the resin at 100˝C for 10 min).  (2-ethylhexyl) ester v a : Components are listed in order of their elution from a HP-5MS column [11]. b : Presence of the compound in the resin, no amount reported [11].

Copal Species, Distribution and Composition
Linares and Bye [15] described extensively how "copaleros" collect the resin. They use a particular knife (quixala or quichala) to make incisions into the bark. They put a leaf of Quercus glaucoides under the cut to isolate the resin from impurities on the bark. To collect the liquid resin, they employ leaves of Agave angustifolia. They collect copal from July to September every year. To avoid killing or damaging the trees, resin is only collected from the same tree every two or three years [15]. These authors reported that the most appreciated species are B. bipinnata (Sessè & Moc. ex DC.) Engl. and B. copallifera (Sessè & Moc. ex DC.) Bullock. Nowadays, painters use copal as a binding medium for paint together with linseed oil.
Most of the phytochemical studies to identify the species used as copal have been done on commercial samples and on archeological Aztec objects [6,16]. "Fresh" resins have a characteristic pine-lemony smell due to volatile terpenes and alkanes such as α-pinene, β-phellandrene, limonene, δ-carene, and heptane [17], while "aged" resins are studied for the triterpenoidic composition of the non-volatile fraction [11]. Pinus resins, are characterized by a large volatile fraction (20%-50%) with monoterpenes predominating over sesquiterpenes while in the non-volatile component diterpene acids with abietane, pimarane, and labdane frameworks are common. Burseraceae resins contain mono-and sesquiterpenes in the volatile fraction and triterpenoids in the non-volatile fraction [1]. Particularly, Protium spp. terpenoids are dominated by αand β-amyrin and Bursera spp. terpenoids contain lupane compounds (e.g., lupeol) [16]. Often the non-volatile fraction of Bursera spp. contains lignans.

Distribution, Synonyms, Common Names, and Primary Essential Oils of Described Bursera Species
A diversity of Bursera species are known to be used as incense, but only a small number are reported by several authors as commercial copal, specifically, B. linanoe, B. copallifera, B. bipinnata, and B. fagaroides. Other important incense sources that are not as commercialized are B. microphylla, B. penicillata, B. simaruba, B. schelechtendalii, and B. excelsa.
Bursera linanoe (La Llave) Rzed., Calderón & Medina (synonyms: B. aloexylon, B. delpechiana, B. longipedunculata, Amyris linaloe, Elaphrium longipedunculatum [13], subg. Elaphrium), also known as Indian lavender tree. This is one of the species most extensively used as copal by the indigenous Mexican people in the past as well as in the present. The XVI century Spanish historian Francisco Hernandez describes this species known to the Aztecs as "Copalcuáuitl", meaning copalli tree, now commonly known as copal blanco [19]. Their drawings of the plant source of this copal also closely resemble live B. linanoe trees, confirming its identity. This species produces one of the most pleasant and fragrant resins and is currently cultivated in India for use in the perfume industry [23]. It is the only Bursera species whose essential oil consists predominantly of linalyl acetate [23,24].
Bursera microphylla A. Gray (synonyms: Elaphrium microphyllum, Terebinthus microphylla, subg. Bursera) is commonly known as elephant tree, torote, torote blanco, copal, or cuajiote colorado and is native to the Sonoran Desert, from southwestern Arizona and southeastern California, to the western Mexican mainland, and Baja California [16,25,26]. The chemistry of this species also varies greatly among geographic locations. The resin acetonic extracts of different samples from two different populations (Guaymas and La Paz) were studied by Mooney and Emboden [27] who identified α-pinene, β-pinene, phellandrene, limonene, cineole, and four unidentified compounds, while Tucker and coll. [28] found that plant samples from Southern Arizona were rich in β-caryophyllene.

Composition of the Triterpenoid Fraction
Triterpenoid of the lupane type are characteristic of Bursera resins, but often ursane and oleanane triterpenoids are also present ( Figure 1). Stacey et al. [6] examined and compared copal resins from different ancient artefacts from the British Museum, botanical specimens from Pinus, Protium and Bursera spp. and commercial samples of copal lágrima, copal negro, copal incienso, copal de piedra and copal blanco, from the market of Tepoztlan, Morelos. They found that the samples of commercial copal blanco, negro and de piedra have similar terpenoid profiles characterized by 3-epi-β-amyrin, 3-epi-α-amyrin, lupeol and α-amyrin, similar to that of a fifty year old B. excelsa sample but with some affinity with B. linanoe. The fresh sample of B. fagaroides var. fagaroides that they examined showed a completely different profile, dominated by oleanonic and ursonic acids. Furthermore, as mentioned above, they found that the commercial sample of copal lágrima has a terpenoid composition resembling Boswellia, due to the presence of boswellic acids.
They analyzed B. bipinnata, B. excelsa, B. copallifera and B. penicillata, but also B. stenophylla as its botanical distinction from B. bipinnata is unclear [21], B. simaruba because it was used as binder in Bonampak murals (Maya) [16] and B. grandifolia because it is phylogenetically related to B. simaruba. In Table 2 we report the phytochemical results for these species. The authors found that the GC-MS profiles of B. bipinnata and B. stenophylla are identical.
Presence of the compound in the resin, no amount reported; b : not found.
They analyzed B. bipinnata, B. excelsa, B. copallifera and B. penicillata, but also B. stenophylla as its botanical distinction from B. bipinnata is unclear [21], B. simaruba because it was used as binder in Bonampak murals (Maya) [16] and B. grandifolia because it is phylogenetically related to B. simaruba. In Table 2 we report the phytochemical results for these species. The authors found that the GC-MS profiles of B. bipinnata and B. stenophylla are identical.
Presence of the compound in the resin, no amount reported; b : not found.
They identified in B. bipinnata all the nine standards and four more unidentified compounds ( Table 2). Studying the triterpenic profile of B. copallifera, Lucero-Gómez et al. found lupeol and lupenone and five other molecules. From the fragmentation pattern in GC-MS analysis, they established the nature of four of them: three urs-12-ene derivatives and one olean-12-ene derivative [16]. In B. excelsa they identified only three of the nine standards: 3-epi-α-and 3-epi-β-amyrins and 3-epi-lupeol. Furthermore, they noted the presence of five other unidentified triterpenoids, three of which had MS fragmentation patterns similar to olean-12-ene type molecules and two of which were amyrones. In B. penicillata, they found a greater variety of triterpenoids than other resins. The only missing standards were α-amyrone and lupenone. Furthermore, they found seven other triterpenoids, three of which were identified as olean-12-ene derivatives ( Table 2). In B. simaruba all of the standards were found except lupenone. Five other triterpenoids were detected in this resin, one of which was identified as an urs-12-ene compound. B. simaruba and B. grandifolia GC profiles are different, as is shown in Table 2.
The phytochemical composition of the non-volatile fraction of B. microphylla fresh resin collected near Hermosillo, Sonora was recently studied by our research group. The methanol extract of the resin was divided by means of solvent with different polarity into two sub-fractions: the hexane sub-fraction (H) and the dichloromethane one (DCM). The H-sub-fraction contained several terpenoids and lignans.
The phytochemical composition of the non-volatile fraction of B. microphylla fresh resin collected near Hermosillo, Sonora was recently studied by our research group. The methanol extract of the resin was divided by means of solvent with different polarity into two sub-fractions: the hexane sub-fraction (H) and the dichloromethane one (DCM). The H-sub-fraction contained several terpenoids and lignans.
The phytochemical composition of the non-volatile fraction of B. microphylla fresh resin collected near Hermosillo, Sonora was recently studied by our research group. The methanol extract of the resin was divided by means of solvent with different polarity into two sub-fractions: the hexane sub-fraction (H) and the dichloromethane one (DCM). The H-sub-fraction contained several terpenoids and lignans.

Composition of the Lignan Fraction
Lignans are phenolic components of foods and medicines that arise from radical coupling of two units of coniferyl alcohol. Lignans can be classified into different groups based on skeleton oxidation and functionalization [34,35]. B. simaruba, B. fagaroides and B. microphylla exudates have been studied for lignan content.
Most of the studies report the lignan content of bark, stem or leaves extracts [36].

Composition of the Lignan Fraction
Lignans are phenolic components of foods and medicines that arise from radical coupling of two units of coniferyl alcohol. Lignans can be classified into different groups based on skeleton oxidation and functionalization [34,35]. B. simaruba, B. fagaroides and B. microphylla exudates have been studied for lignan content. Most of the studies report the lignan content of bark, stem or leaves extracts [36]. Velazquez-Jimenez et al. [37] isolated from B. fagaroides resin two aryltetraline lignans ((−)-deoxypodophyllotoxin, (−)-morelensin) and two dibenzylbutirolactone lignans ((−)-yatein and (−)-5′-desmethoxyyatein). The authors determined the absolute configuration of these compounds by comparison of the vibrational circular dichroism spectra of known podophyllotoxin and desoxypodophyllotoxin with those obtained by density functional theory calculations. Other diarylbutane lignans were isolated by Morales-Serna et al. [38] from the chloroform extract of B. fagaroides resin: 9-acetyl-9′pentadecanoyl-dihydroclusin, 2,3-demethoxy-secoisolintetralin monoacetate, dihydroclusin monoacetate, together with previously known 2,3-demethoxysecoisolintetralin diacetate and dihydroclusin diacetate ( Figure 5).     It is interesting to note that lignans isolated from copal, with the exception of burseran, belong to the aryltetraline and dibenzylbutane groups. Koulman studied the biosynthesis of Bursera lignans from matairesinol and he classified them in four different groups (Figure 8) [36]. From his studies on dry leaves, Koulman noted that groups 1 and 2 lignans are present in subg. Bursera (B. fagaroides and B. microphylla) while groups 3 and 4 are in subg . Elaphrium (B. bipinnata, B. copallifera, B. cuneata,  B. excelsa and B. penicillata). The few studies on lignans isolated from Bursera resins, are in agreement with these results. Lactone lignans isolated from B. fagaroides and B. microphylla (both in subg Bursera) belong to group 1.   It is interesting to note that lignans isolated from copal, with the exception of burseran, belong to the aryltetraline and dibenzylbutane groups. Koulman studied the biosynthesis of Bursera lignans from matairesinol and he classified them in four different groups (Figure 8) [36]. From his studies on dry leaves, Koulman noted that groups 1 and 2 lignans are present in subg. Bursera (B. fagaroides and B. microphylla) while groups 3 and 4 are in subg. Elaphrium (B. bipinnata, B. copallifera, B. cuneata,  B. excelsa and B. penicillata). The few studies on lignans isolated from Bursera resins, are in agreement with these results. Lactone lignans isolated from B. fagaroides and B. microphylla (both in subg Bursera) belong to group 1. It is interesting to note that lignans isolated from copal, with the exception of burseran, belong to the aryltetraline and dibenzylbutane groups. Koulman studied the biosynthesis of Bursera lignans from matairesinol and he classified them in four different groups (Figure 8) [36].

Biological Activities of Copal.
Case and Orta-Amaro [3,41] described many of the ancient and traditional uses of copal. Mesoamerican people used copal for different purposes. First of all, it was considered to be food for the gods, but it was also used as incense used during ceremonies, as a binder mixed with pigments for painting, for the decoration of murals and for the preparation of holy artifact. "Copal served as the "flesh" of idols with a wooden skeleton that were further covered with a rubber skin" [3]. Copal smoke was used to cure headache and to clean the body after being exposed to sick people [19]. Copal ground and dissolved in water was used by the Nahua people to treat diarrhea, as an anti-inflammatory poultice [41], to plug tooth cavities (Nahua and Maya), and to treat pneumonia. Bursera copallifera was used against uterine diseases. Bursera bipinnata has been used to treat wounds and B. simaruba has been used to treat fever and chicken pox [3]. The Seri Pharmacopoeia reports the use of B. microphylla resin for sore throats, headache, and for wound healing [42]. In Oaxaca city, copal of B. fagaroides is used to prepare infusions to treat stomach problems and as an anti-inflammatory [38].
Copal was, and still is, primarily used as folk medicine and only a few scientific papers are found in the literature that have investigated its biological activity. Bursera bipinnata copal was studied for its film-forming potential and its potential use as coating material for sustained release and colon-targeted drug delivery [43]. Velazquez-Jimenez et al. prepared an ethanol extract of the dry exudate of B. fagaroides and found it cytotoxic, in a concentration-dependent manner, against HT-29 (human colorectal adenocarcinoma) cells with IC50 values of 0.40 and 0.41 μg/mL after 48 and 72 h, respectively [37]. As already mentioned, these authors isolated from this extract four podophyllotoxin related lignans from this extract [37]. The methanol extract of B. microphylla was studied for its cytotoxic activity against human cancer cell lines: A549 (lung cancer) (IC50 53.77 μg/mL) andHeLa (cervix cancer) (IC50 13.85 μg/mL), and against the murine cell lines M12.C3.F6 (B cell lymphoma) (IC50 26.00 μg/mL). Following these encouraging preliminary results, the new and the already known compounds isolated by hexane fraction were tested for their cytotoxic activity against three human cancer cell lines, namely, A549, HeLa, and PC-3 (prostate cancer), and against the murine cell lines M12.C3.F6 and RAW264.7 (macrophages transformed by the virus Abelson leukemia). Malabricatrienone, malabaricatrienol and microphyllanin (Figures 2 and 3) were found to be inactive, and among the known compounds, only dihydroclusin diacetate was shown to be active against murine cell line M12.C3.F3 (IC50 2.5 μM), while ariensin, burseran, and dihydroclusin diacetate (

Biological Activities of Copal.
Case and Orta-Amaro [3,41] described many of the ancient and traditional uses of copal. Mesoamerican people used copal for different purposes. First of all, it was considered to be food for the gods, but it was also used as incense used during ceremonies, as a binder mixed with pigments for painting, for the decoration of murals and for the preparation of holy artifact. "Copal served as the "flesh" of idols with a wooden skeleton that were further covered with a rubber skin" [3]. Copal smoke was used to cure headache and to clean the body after being exposed to sick people [19]. Copal ground and dissolved in water was used by the Nahua people to treat diarrhea, as an anti-inflammatory poultice [41], to plug tooth cavities (Nahua and Maya), and to treat pneumonia. Bursera copallifera was used against uterine diseases. Bursera bipinnata has been used to treat wounds and B. simaruba has been used to treat fever and chicken pox [3]. The Seri Pharmacopoeia reports the use of B. microphylla resin for sore throats, headache, and for wound healing [42]. In Oaxaca city, copal of B. fagaroides is used to prepare infusions to treat stomach problems and as an anti-inflammatory [38].
Copal was, and still is, primarily used as folk medicine and only a few scientific papers are found in the literature that have investigated its biological activity. Bursera bipinnata copal was studied for its film-forming potential and its potential use as coating material for sustained release and colon-targeted drug delivery [43]. Velazquez-Jimenez et al. prepared an ethanol extract of the dry exudate of B. fagaroides and found it cytotoxic, in a concentration-dependent manner, against HT-29 (human colorectal adenocarcinoma) cells with IC 50 values of 0.40 and 0.41 µg/mL after 48 and 72 h, respectively [37]. As already mentioned, these authors isolated from this extract four podophyllotoxin related lignans from this extract [37]. The methanol extract of B. microphylla was studied for its cytotoxic activity against human cancer cell lines: A549 (lung cancer) (IC 50 53.77 µg/mL) andHeLa (cervix cancer) (IC 50 13.85 µg/mL), and against the murine cell lines M12.C3.F6 (B cell lymphoma) (IC 50 26.00 µg/mL). Following these encouraging preliminary results, the new and the already known compounds isolated by hexane fraction were tested for their cytotoxic activity against three human cancer cell lines, namely, A549, HeLa, and PC-3 (prostate cancer), and against the murine cell lines M12.C3.F6 and RAW264.7 (macrophages transformed by the virus Abelson leukemia). Malabricatrienone, malabaricatrienol and microphyllanin (Figures 2  and 3) were found to be inactive, and among the known compounds, only dihydroclusin diacetate was shown to be active against murine cell line M12.C3.F3 (IC 50 2.5 µM), while ariensin, burseran, and dihydroclusin diacetate ( Figure 5) were active against the RAW246.7 murine cell line (IC 50 9.8, 0.4, and 0.2 µM, respectively). Betulonic acid (Figure 2) was shown to be active against all the tested lines (IC 50 : M12.C3.F3 = 13.2 µM, A549 = 12.6 mM, HeLa = 13.6 µM, RAW 264.7 = 10.2 µM, PC-3 = 18.6 µM) [31].
Although few studies have been reported on the biological activities of Bursera copal, several of the isolated compounds have been studied. Many terpenoids and lignans isolated from Bursera copal have been studied and several reviews on their biological activity have been published. For example biological properties of lupeol, αand β-amyrins and lignans have been recently reviewed [44][45][46].

Conclusions
Our analysis of the literature showed that in Mesoamerica the term "copal" currently does not have an unequivocal botanical association and that, despite continued widespread use, few data are available on the analytical composition of these resins. Due to the extensive studies of historical artifacts, most of the research efforts have been conducted on the triterpenoid fraction, while a limited number of studies are reported about the volatile fraction composition. Furthermore, deeper studies have to be made to validate the biological and pharmacological properties of these resins that are commonly used in ethnopharmacology.