Introduction

Burseraceae family is distributed in tropical and subtropical regions around the world. The taxonomic classification of Burseraceae has been a long and unsatisfactory process; several plant characteristic (flower, fruit and seed anatomies, germination, etc.) has been used as basis to clarify the subdivision of this family^1,2. Actually, Burseraceae family is formed by 18 genres and more than 600 species; nine of these genres are only found in American continent³.

Bursera is one of Burseraceae genus found only in American continent and it contains more than 100 species distributed from the southern United State to Peru and the Caribbean². Actually, the genus is divided in three subgenus: Bursera, Elaphrium and Buntingia⁴. In Venezuela, there are reported 6 species of Bursera, 3 of them are endemic of northern South America and Caribbean.

Traditionally, species of Bursera genus has been used by some populations in the treatment of several illness and symptoms, such as headache, toothache, uterine and urinary diseases, fever, rheumatism, bronchitis, eczema, etc^3,5,6,7.

Species belonging to this genus are usually rich in essential oils and resins which are usually composed by terpenoids⁸. Regarding the diverse reports for the chemical composition of essentials oils and resins from Bursera species, it is found a significant variation in number, identity and individual relative amounts of terpenoids described in the analyzed species even in populations of the same species.

As part of our phytochemical study of Bursera genus, three species found in Mérida state (Venezuela), B. glabra Jacq., B. inversa Daly and B. simaruba L. Sarg., were studied. In this article we describe the identification by GC-MS of compounds presents in resins from the three selected species and the chemical composition of essential oil of fruits from B. simaruba L. Sarg.

Experimental

General experimental procedures

The GC-MS analysis were carried out on a Hewlett Packard GC-MS system, Model 5973, fitted with a 30 m long, crosslinked 5 % phenylmethyl siloxane (HP-5MS, Hewlett Packard, USA) fused-silica column (0.25 mm, film thickness 0.25 μm). Source temperature 230 °C; quadrupole temperature 150 °C; carrier gas helium, adjusted to a linear velocity of 34 m/s; ionization energy, 70 eV; scan range 40-500 amu; 3.9 scans/s. The injected volume was 1.0 μL. The initial oven temperature was 100 °C and then raised to 300 °C at 5 °C/min for resins, essentials oils and fatty acid methyl esters. The final temperature was maintained for 20 min. A Hewlett-Packard ALS injector was used with split ratio 1:100. Identification of the resin components was supported on the Wiley MS Data Library (6th edition), followed by comparison of MS data with published literature9,10. Kováts retention indices (KI) were determined relative to the retention times of a series of n-paraffin hydrocarbons (C7-C22)^9,10.

Plant material

The samples of three Bursera species were collected in Méri- da (Venezuela):

1. B. simaruba (L.) Sarg.:

   Location A: collected on July 2015 at 1100 m.a.s.l. in the xerophytic zone between Ejido and Las Gonzales, in the highway to Los Guáimaros (Mérida State). Coordinates: 8º32’4.7ʺ N; 71º15’34.2ʺ W. Samples of two different specimens were collected (A1 and A2)

   Location B: collected on July 2015 at 1050 m.a.s.l. in the Botanic Garden “Ing. Carlos Liscano” (San Juan de Lagunillas, Mérida State). Coordinates: 08º30’38.0” N; 71º21’98.5” W. Samples of three different specimens were collected (B1, B2 and B3) Location C: collected on October 2015 at 710 m.a.s.l. in rural highway between Estanques and Mesa Bolivar (Mérida State). Coordinates: 8º27’52” N; 71º32’44” W.

2. B. glabra (Jacq.): Collected in blooming period on February 2016 at 1900 m.a.s.l. in highway to El Morro, between El Morro’s bridge and the detour to El Mocaz (Mérida State). Coordinates: 8º27’00.7” N; 71º11’09.1” W.

3. B. inversa Daly: Collected in fruit period on February 2016 at 36 m.a.s.l. in El Vigía (Mérida state), Bolívar Avenue, in the way to Santa Bárbara del Zulia. Coordinates: 8º38’24.1” N; 71º40’3.9” W.

   The samples identification was carried on by professors José Guevara and Enrique Gámez from the Faculty of Forest and Environmental Sciences (Universidad de Los Andes), professor Mercedes Castro from Agronomy Faculty (Universidad Central de Venezuela) and staff of the Botanic Garden “Ing. Carlos Liscano”.

Resin samples collection and treatment

To collect resin samples, a cut was made over the plant, approximately at one meter of height over the ground, with a sterile surgical knife to remove a bark piece of 50 cm2 and 2-4 mm of thickness. Then, after 2-3 min, the resin started to appear. The resin was collected in opaque test tubes and stored at low temperatures until the analysis to avoid the degradation and aging of the resin.

For the GC-MS analysis, around 5 mg of resin was solubilized in 10 ml of hexane and few drops of dichloromethane, then it was shake until the resin was completed dissolved. The injected volume was 1.0 μl.

Fruit samples collection and treatment

B. simaruba fruits (200 g) were cut to separate the fleshy mesocarp (160 g) and the osseous endocarp (40 g).

Essential oil from mesocarps: The mesocarp was cut in small pieces and then was submitted to hydrodestillation by 3 hours with a Clevenger tramp to obtain 4 ml of essential oil. This was dried with Na2SO4 anhydride and kept under atmosphere of N2 at 4 ºC.

For the GC-MS analysis the essential oil was solubilized in hexane. The injected volume was 1.0 μL.

Fatty acids from endocarps: The endocarp was dried at 40 ºC by 24 hours. Then it was powdered and submitted to Soxhlet extraction with hexane at 65 ºC by 48 hours to obtain a yellowish oil. This oil was extracted with NaOH 10% to yield 410 mg of a mixture of fatty acids. After, the mixture of fatty acids was treated with diazomethane to obtain the methyl esters.

For the GC-MS analysis essential oil was solubilized in ethyl ether. The injected volume was 1.0 μl.

Results and discussion

The resin samples obtained from the three Bursera species were analyzed by GC-MS in order to identify the volatile compounds of the samples. The GC-MS analysis yields a total of twenty-nine well-known monoterpenes and sesquiterpenes. The compounds were identified by comparison of spectrometry data with literature values. The table 1 shows the compounds and its relative amount (weight percent, % w/w) found in every resin sample.

Results in table 1 showed ignificant difference in the compounds found in resins from B. inversa, B. glabra and B. simaruba. The difference in its chemical composition could be an indicator that confirm the subdivision of Bursera genus in three subgenus proposed by Castro-Laportte4, where B. inversa, B. glabra and B. simaruba are classified inside the Buntingia, Elaphrium and Bursera subgenus, respectively.

In B. simaruba resins were identified eighteen components in the six samples analyzed which corre-spond to 90.3-95.7 % of the total resins. These resins are dominated by monoterpenes. All resin samples has a higher amount of monoterpenes (59-90 %) than sesquiterpenes (3-33 %) where the α-pinene is the major compound (52-66 %). The results shows that resin samples of B. simaruba could be separated in two groups based in the proportion of monoterpenes and sesquiterpenes found in the samples. One group has a monoterpene/sesquiterpene ratio of approximately 90/3 and the other a 60/30 ratio. Samples having ~90 % of monoterpenes (B2, B3 and C) contain a significant amount of monoterpene α-phellandrene (24-31 %) which was not observed in other group. This last one group showed an increase in the amount of some sesquiterpenes as germacrene D (11- 18 %), α-copaene (5-6 %) in A1 and A2, β-bourbonene (4.5 %) in A1, α-cubebene (5.1 %) in A2 and β-caryophyllene (14.6 %) in B1.

There are previous phytochemical analysis of essentials oils from B. fagaroides¹¹, B. hollicki¹², B. lunani¹³, B. morelensis¹⁴ and B. schlechtendalii8 species, which belongs to Bursera subgenus. These studies showed that monoterpenes α-pinene and α-phellandrene are usually major compounds of essentials oils from species classified in this subgenus. The comparison between obtained results for resin of B. simaruba and the chemical composition of leaves, fruits and bark essential oils from the same plant found in Jamaica¹⁵ showed that essential oils of both species have a similar composition with α- pinene as the major component. The mainly difference between Venezuelan and Jamaican species is the presence of germacrene D in all B. simaruba samples collected in Mérida (Venezuela). Since the resin composition from Jamaican and Venezuelan species of B. simaruba are similar, it seems that identity of resin components is principally inherent to the specie and external factors (as region, climate, soil, etc.) could affect the relative amount of these components, which could explain the presence of germacrene D in Venezuelan species. However, it is necessary a detailed research under controlled conditions to determine the accurate causes of the observed chemical composition variations.

Resin from B. glabra is constituted by eight components which correspond to 94.1 % of total resin. This resin is composed mainly by monoterpenes (90.5 %) with a proportion of monoterpenes and sequiterpenes similar to samples B1, B2 and C of B. simaruba. However, for the B. glabra resin the main compounds are limonene and cis-ocymene (77.6 % and 7.9 %, respectively) and the α-pinene is not reported. Despite of B. glabra and B. simaruba resins have a similar monoterpenes/sesquiterpenes proportion, the chemical composition of B. glabra resin is very different to B. simaruba resins, and α-phellandrene is the only one compound in common for the resins of both species.

The analysis of the volatile compounds for other species of Elaphrium subgenus revealed the monoterpenes limonene and cis-ocimene as major compounds in the essentials oils and resins from B. graveolens^16,17,18 and B. tomentosa¹⁹.

The specie B. inversa is the only specie classified inside Buntingia subgenus and there is a great difference in chemical composition between B. inversa resin and the resins from the other species studied in this work. In B. inversa resin, 10 components were identified which constituted 87.4 % of total resin. The predominant compounds are sesquiterpenes and represent the 86.4 % of the composition, with a proportion of 86/1 of sesquiterpenes/monoterpenes. The principal compounds are α-humulene (27.7 %), β-caryophyllene (22.1 %) and germacrene B (16.3 %).

The comparison between B. inversa resins and B. glabra and B. simaruba resins showed that B. inversa has some compounds in common with B. glabra and B. simaruba. However, the main ompounds found in resin of B. glabra and B. simaruba were not located in B. inversa resin or their relatives amounts are no substantial (<5 %).

The B. simaruba fruits were separated in mesocarps and endocarps in order to determinate their essential oil chemical compositions. Results are listed in Table 2. The analysis allowed the identification of thirteen compounds in mesocarps and four components in endocarps. The components identified in mesocarps and endocarps correspond to 99.5 % and 87.3 % of total oils, respectively. The endocarps are composed principally, as it was expected, by fatty acids (determined as methyl esters) with the linoleic acid (28.5 %), palmitic acid (25.2 %) and oleic acid (24.7 %) as major components while for the mesocarps its essential oil is composed only by monoterpenes and the major compounds are sabinene (59.2 %), terpinen-4-ol (12.3 %) and α-pinene (7.6 %).

The composition of the mesocarp essential oil is very similar in terpenoid identity to essential oil from Jamaican B. simaruba fruits¹⁵. However, the relative amounts and major components are slightly different. In Jamaican fruits, the major compound are α-pinene (28 %), β-pinene (24 %) and terpinen-4-ol (13 %), whereas the sabinene is present in a minor amount (8 %). This difference in the amount relative of each compound could be explained if, as mentioned above, the environment of the analyzed specie has influence in relative amount of secondary metabolites produced by the plant.

Conclusions

The analysis of resins from B. simaruba, B. glabra and B. inversa and essential oil of B. simaruba fruits allowed a better knnowledge of the chemical composition of this species. Since there are not previous reports for B. glabra and B. inversa resins, the identification of the resin components is a great contribution to increase the phytochemical knowledge of Bursera species.

Results of B. inversa resins are remarkable due to higher amount of sesquiterpenes. The other studied species showed a major proportion de monoterpenes than sesquiterpenes.

Since results from chemical analysis of B. simaruba and B. glabra resins and essential oil of B. simaruba fruits are whole consistent with the data reported to other species belonging to the same subgenus, a detailed research of the secondary metabolites in Bursera species could showed the presence of chemotaxonomic markers specific for each Bursera specie. In addition, further studies under controlled conditions will allow determine a relationship between environment and relative amount essential oils components.

References

1 J Rzedowski, RM Lemus, GC Rzedowski. Las especies de Bursera (Burseraceae) en la cuenca superior del río Papaloapan (México). Acta Botánica Mex. 66, 23–151 (2004).

2 JX Becerra, K Noge, S Olivier, DL Venable. The monophyly of Bursera and its impact for divergence times of Burseraceae. Taxon 61(2), 333–343 (2012).

3 ME De la Cerda-Lemus. La familia Burseraceae en el estado de Aguascalientes, México. Acta Botánica Mex. 94(1), 1–25 (2011).

4 M Castro-Laportte. Propuesta de un nuevo subgénero para el género Bursera Jacq. Ex l. (Burseraceae), con comentarios sobre Bursera inversa Daly. Ernstia 23(1), 67–82 (2013).

5 SA Mitchell, MH Ahmad. A review of medicinal plant research at the University of the West Indies, Jamaica, 1948 – 2001. West Indian Med. J. 55(876), 243–269 (2006).

6 JX Becerra, K Noge. The Mexican roots of the Indian lavender tree. Acta Botánica Mex. 91(1), 27–36 (2010).

7 RJ Case, AO Tucker, MJ MacIarello, KA Wheeler. Chemistry and ethnobotany of commercial incense copals, copal blanco, Copal Oro, and Copal Negro, of North America. Econ. Bot. 57(2), 189–202 (2003).

8 PH Evans, JX Becerra, DL Venable, WS Bowers. Chemical analysis of squirt-gun defense in Bursera and counterdefense by chrysomelid beetles. J. Chem. Ecol. 26(3), 745–754 (2000).

9 RP Adams. Identification of essential oils components by gas chromatography/mass spectroscopy. Allured Publishing: Illinois, USA (2007).

10 NW Davies. Gas chromatographic retention indices of monoterpenes and sesquiterpenes on methyl silicon and Carbowax 20M phases. J. Chromatogr. A 503, 1–24 (1990).

11 K Noge, JX Becerra. Germacrene D, a common sesquiterpene in the genus Bursera (Burseraceae). Molecules 14(12), 5289–5297 (2009).

12 GAO Junor, RB . Porter, T Yee, LA . Williams. Chemical composition and insecticidal activity of the essential oils from Bursera hollickii (Britton) found in Jamaica. J. Essent. Oil Res. 20(6), 560–565 (2008).

13 GAO Junor, RBR Porter, TH Yee. Chemical composition of essential oils from the aerial parts of Jamaican Bursera lunanii Spreng. J. Essent. Oil Res. 22(6), 602–605 (2010).

14 CA Carrera-Martínez, R Rosas-López, MA Rodríguez-Monroy, MM Canales-Martínez, A Román-Guerrero, R Jiménez- Alvarado. Chemical composition and in vivo anti-inflammatory activity of Bursera morelensis Ramírez essential oil. J. Essent. Oil Bear. Plants 17(5), 758–768 (2014).

15 GAO Junor, RBR Porter, TH Yee. The chemical composition of the essential oils from the leaves, bark and fruits of Bursera simaruba (L) Sarg. from Jamaica. J. Essent. Oil Res. 20(5), 426–429 (2008).

16 R Carmona, CE Quijano-Celís, JA Pino. Leaf oil composition of Bursera graveolens (Kunth) Triana et Planch. J. Essent. Oil Res. 21(5), 387–389 (2009).

17 DG Young, S Chao, H Casablanca, MC Bertrand, D Minga. Essential oil of Bursera graveolens (Kunth) Triana et Planch from Ecuador. J. Essent. Oil Res. 19(6), 525–526 (2007).

18 A Muñoz-Acevedo, A Serrano-Uribe, XJ Parra-Navas, LA Olivares-Escobar, ME Niño-Porras. Análisis multivariable y variabilidad química de los metabolitos volátiles presentes en las partes aéreas y la resina de Bursera graveolens (Kunth) Triana & Planch. de Soledad (Atlántico, Colombia). Boletín Latinoam. y del Caribe Plantas Med. y Aromáticas 12(3), 322–337 (2013).

19 J Moreno, R Aparicio, J Velasco, LB Rojas, A Usubillaga, M Lue-Meru. Chemical composition and antibacterial activity of the essential oil from fruits of Bursera tomentosa. Nat. Prod. Commun. 5(2), 311–313 (2010).