Composition and Biological Activity of Essential Oils from Protium confusum Ana I. Santanaa,b, Roser Vilac, Alex Espinosaa, Dionisio Olmedoa, Mahabir P. Guptaa and Salvador Cañigueralc,* aCentro de Investigaciones Farmacognósticas de la Flora Panameña (CIFLORPAN), Facultad de Farmacia, Universidad de Panamá, Apartado 0824 00172, Panamá, Republic of Panama bDepartamento de Química Orgánica, Facultad de Ciencias Naturales, Exactas y Tecnología, Universidad de Panamá, Apartado 0824 00178, Panama, Republic of Panama cUnitat de Farmacologia i Farmacognòsia, Facultat de Farmàcia, Universitat de Barcelona, Avda. Diagonal, 643, E-08028 Barcelona, Spain s.canigueral@ub.edu Received: February 26th, 2009; Accepted: April 13th, 2009 The composition and biological activity of the essential oils from leaves, fruits, stems and bark of Protium confusum are reported for the first time. Forty-six to sixty-three constituents were identified ranging from 73.8% to 98.5% of the samples. Limonene (60.2%) was the main component in the fruit oil, whereas spathulenol (19.3%), β-caryophyllene oxide (14.1%) and β-caryophyllene (8.0%) reached the highest percentages in the oil from leaves. The volatile oils from bark and stems showed the same major constituents: p-cymen-8-ol (14.4% and 6.1%, respectively), spathulenol (9.5% and 9.0% respectively), and hexadecanoic acid (8.4% and 7.8%, respectively). The composition of the essential oils differed significantly from those of previously investigated Protium species. Three out of the four samples exhibited antibacterial activity against Staphylococcus aureus and Mycobacterium smegmatis, the one from leaves being the most active, with MIC values of 62.5 μg/mL in both cases. No activity against Candida albicans was detected. The bark oil showed the highest larvicidal activity against Aedes aegypti (LC100 = 125 μg/mL). Keywords: Protium confusum, essential oils, limonene, spathulenol, β-caryophyllene oxide, p-cymen-8-ol, antibacterial activity, larvicidal activity. The genus Protium is one of the largest and most heterogeneous of the Burseraceae family, comprising about 135 species, which predominate in the neotropics. Many of them are attractive commercial botanical sources of aromatic products, such as oleoresins and essential oils, which have been used since ancient times as important therapeutic agents in folk medicine for their wound healing, anti-inflammatory, tonic, stimulant, analgesic and expectorant properties [1,2]. The decoction of the bark of P. panamense is used in Nicaragua, both orally and topically, for aches and pains, as a digestive and for expelling worms [3]. Popularly, the resins of Protium spp., which are an important source of copal [4], are burned and used as an insect repellent and as incense [1]. Some of them also provide valuable essential oils to the perfumery industry.

Within the framework of our ongoing research on aromatic flora from Panama, the present work deals with the chemical composition and the biological activity of the essential oils from stem bark, stems, leaves and fruits of Protium confusum (Rose) Pittier (synonyms P. correae D.M. Porter, P. inconforme Pittier, P. schippii Lundell, and Icica confusa Rose) [5], which is commonly known in Panama as copal, copá, chutra, and alcanfor. It is a tree 5-15 m in height with alternate imparipinnated leaves and green to yellowish aromatic flowers. The fruits are ellipsoid green capsules changing to red on maturity. The plant grows between low and medium elevations in humid tropical climates in Mesoamerica. The detachment of any part of the plant yields a transparent, colorless and aromatic resinous exudate.

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1402 Natural Product Communications Vol. 4 (10) 2009 Santana et al.

Table 1: Constituents of the essential oils from different plant parts of Protium confusum.

%d Constituentsa RI-CWb RI-SEc Fruits Leaves Stems Bark

α-Pinene 112 208 3.9e,f,g - - - Camphene 129 213 0.1e,f,g - - - β-Pinene 150 225 1.0e,f,g - - - Sabinene 158 - te,f,g - - - 3-Carene 175 243 9.4e,f,g - - - Myrcene 180 232 1.0e,f,g - - - α-Terpinene 200 237 4.2e,f,g - - 0.2e,g Limonene 211 255 60.2e,f,g te,g te,g 0.4e,f,g β-Phellandrene 211 - 0.3e,f,g - - - cis-Ocimene 221 - te,f,g - - - γ-Terpinene 226 266 1.6e,f,g - - - p-Cymene 239 244 6.6e,f,g 0.1e,g 0.7e,f 2.2e,f,g Terpinolene 243 280 0.4e,f,g te,g 0.2e,g - α-p-Dimethylstyrene 321 277 0.3e,f,g - 0.9e,f,g 0.6e,f,g α-Cubebene 322 415 0.1e,g 0.1e,f,g 0.1e,f,g 0.4e,g cis-Limonene oxide 323 300 0.1e,f,g - - - trans-Limonene oxide 329 - 0.1e,f - - - α-Ylangene 340 427 0.3e,f,g 0.5e,f,g 0.3e,f,g 0.5e,f,g α-Copaene 344 425 0.1e,f,g 0.5e,f,g 0.5e,f,g 0.5e,g Campholenal 346 - - - - 0.6e,f α-Gurjunene 367 437 te,g 0.3e,f - 1.4e,f,g Linalool 375 285 - - - 0.5e,f,g Pinocarvone 380 309 - - 0.4e,f,g 0.8e,f,g β-Elemene 395 432 te,f,g 0.9e,g 0.2e,f,g 0.8e,f,g β-Caryophyllene 400 447 0.1e,f,g 8.0e,f,g 0.7e,f,g 0.5e,g Terpinen-4-ol 402 322 0.2e,f,g - te,f,g 0.8e,f cis-Dihydrocarvone 408 - 0.2e,f - - 0.6e,f Myrtenal 409 333 0.1e,g 0.3e,g 1.2e,f,g 1.8e,f,g cis-Verbenol 413 - 0.1e,f - - - allo-Aromadendrene 418 - - 0.3e,f - - trans-Pinocarveol 424 304 - 0.1e,g 1.2e,f,g 2.2e,f,g Aromadendrene 430 476 0.1e,f,g - - te,g α-Humulene 436 461 0.1e,g 4.7e,f,g 0.3e,f,g 0.8e,g Neral 441 - 0.1e,f - - - α-Terpineol 446 330 4.2e,f,g 1.2e,f,g te,g 0.3e,g Verbenone 448 336 0.2e,g - 5.2e,f,g 4.0e,f,g γ-Muurolene 450 490 0.1e,f,g 0.3e,f,g 0.8e,f,g - β-Selinene 456 478 0.1e,f 4.0e,f,g 1.3e,f,g 1.4e,f,g α-Selinene 458 483 0.1e,f,g 3.1e,f,g - te,g Carvone 462 348 0.2e,f,g 0.1e,g 1.2e,f,g 4.0e,f,g δ-Cadinene 473 495 0.2e,f,g 1.3e,f,g 0.6e,f,g 1.0e,f,g γ-Cadinene 480 490 - - - 0.7e,f,g Methylacetophenone 481 318 0.1e,f,g - 3.0e,f,g - Cuminaldehyde 483 347 - - 1.4e,f,g 1.7e,f,g α-Curcumene 486 - - te,f - - Myrtenol 490 336 - - 1.1e,f,g 1.3e,f cis-Carveol 500 350 - - - 2.0e,f,g Germacrene B 505 - te,f,g 0.2e,f - - Calamenene 512 500 te,f 0.2e,f 0.5e,f,g - trans-Carveol 513 345 0.1e,f 0.1e,f,g 1.0e,f,g 2.1e,f,g Thymyl acetate 518 - 0.1e,f - - - p-Cymen-8-ol 520 324 0.3e,f,g 1.2e,f,g 14.4e,f,g 6.1e,f,g α-Calacorene 548 500 0.1e,f 1.1e,f,g 1.0e,f,g 0.9e,f,g β-Ionone 561 - - 0.2e,f - - β-Caryophyllene oxide 577 528 te,f 14.1e,f,g 3.8e,f,g 4.1e,f,g endo-Bourbonanol 585 531 - 0.3e,f - 1.9e,f,g Cubenol 616 546 0.1e,f,g 1.3e,f,g 0.6e,f,g 2.3e,f,g Globulol 626 544 - 2.0e,f,g - - Viridiflorol 627 530 - 2.1e,f,g 0.6e,f 0.3e,f Cuminol 638 381 - 0.5e,f 0.8e,f,g - Spathulenol 648 525 0.2e,f,g 19.3e,f,g 9.5e,f,g 9.0e,f,g 10-epi-Cadinol 676 - - 1.0e,f - 0.6e,f Thymol 686 378 te,f,g 0.1e,f,g 1.2e,f,g 0.5e,f,g T-Muurolol 686 - te,f - - - α-Bisabolol 690 573 - 1.4e,f,g - - Carvacrol 700 - te,f - 0.9e,f 1.1e,f,g Isospathulenol 707 549 - 2.1e,f,g - 1.2e,f,g α-Cadinol 708 558 te,f,g 2.1e,f,g 1.9e,f,g 2.8e,f,g Fitol >800 - - 0.2e,f te,f - Tetradecanoic acid >800 623 - - 1.9e,f,g 1.3e,f,g Pentadecanoic acid >800 675 - te,f 1.4e,f,g 0.8e,f,g Hexadecanoic acid >800 728 0.3e,f,g 2.1e,f,g 8.4e,f,g 7.8e,f,g

Essential oils of Protium confusum Natural Product Communications Vol. 4 (10) 2009 1403

%d Constituentsa RI-CWb RI-SEc Fruits Leaves Stems Bark

Linoleic acid >800 800 - 0.2e,f,g 4.9e,f,g 1.5e,f,g Tricyclene - 205 0.1e,g - - - trans-Ocimene - 261 0.1e,g - - - Dihydrolinalool - 292 - - 0.2e,g - Bornyl acetate - 376 0.1e,g 0.2e,g - 0.3e,g Longifolene - 453 te,g - - - α-Santalene - 455 - 0.5e,g - - β-Cubebene - 475 0.1e,g 0.4e,g - - Monoterpene hydrocarbons 89.3 0.2 1.8 3.4 Oxygenated monoterpenes 6.2 3.8 30.3 30.7 Sesquiterpene hydrocarbons 1.7 26.4 6.3 9.0 Oxygenated sesquiterpenes 0.4 45.7 16.4 22.2 Others 0.4 2.7 19.6 11.4 Total identified 98.0 78.8 74.4 76.7

a Components are listed in increasing order according to their retention indices on SupelcowaxTM 10, except the last seven constituents, which were only detected on methylsilicone.

b RI-CW: Retention indices in SupelcowaxTM10. c RI-SE: Retention indices in methylsilicone (SE-30). d t: traces (≤ 0.05). e,f,g: Identification methods: e GC-MS, f Retention index in SupelcowaxTM10, g Retention index in methylsilicone.

In Panama, the resin of P. confusum is used in folk medicine for the treatment of colds, asthma and headache, as an anthelmintic and for its wound healing properties [6]. Until now, neither data on the chemical constituents nor the biological activity of the resin or extracts of this species are available in the scientific literature, although those of other Protium spp. have been previously studied [7-15]. The essential oils from the oleoresins of several Protium spp. have been shown to possess antimicrobial, anti-inflammatory, antinociceptive, antioxidant, anti-tumor, acaricidal, molluscicidal and cercaricidal activities [2,16-21]. Some of these activities have also been reported for the volatile oils from other parts of the plants, mainly leaves and fruits [1,2]. The air-dried fruits, leaves, bark and stems of P. confusum gave by hydrodistillation essential oils with yields of 0.65%, 0.13%, 0.10% and 0.05%, respectively. The results obtained are shown in Table 1. In the oil from fruits, fifty-six constituents were identified, representing 98% of the total sample. Monoterpene hydrocarbons (89.3%) were the main constituents, limonene (60.2%) being the major one, followed by 3-carene (9.4%) and p-cymene (6.6%). In contrast, the leaf oil was characterized by a high percentage of sesquiterpenes (ca. 72%), mainly oxygenated (45.7% of the oil), among which spathulenol (19.3%), β-caryophyllene oxide (14.1%) and β-caryophyllene (8.0%) reached the highest percentages. More than 78% of the oil (45 components) was identified.

The oils from stems and bark showed a similar composition pattern, characterized by a high content of oxygenated compounds (66.3% and 64.3%, respectively), well differentiated from the essential oils from fruits and leaves. Forty-one and forty-seven compounds were identified, respectively in these samples, representing 74.4% and 76.7% of the oils. p-Cymen-8-ol (14.4% and 6.1%), spathulenol (9.5% and 9.0%) and hexadecanoic acid (8.4% and 7.8%) were found to be the major constituents. Several papers on the chemical composition of the essential oil from the resins of Protium spp. can be found in the literature, but none dealing with the volatile oils from aerial parts of the plants, such as fruits, bark, branches and leaves. Among them, the oils from leaves of different Protium spp. have been reported by different authors to contain mainly sesquiterpenes, such as caryophyllene derivatives, and trans-isolongifolanone or β-elemene in P. heptaphyllum [1,22], germacrene-type sesquiterpenes, together with α-copaene, γ-elemene and δ-cadinene in P. icicariba [23], α- and β-selinene in P. strumosum, and β-caryophyllene, δ-cadinene and α-humulene in P. grandifolium [2]. Furthermore, in the leaf oil of some species, monoterpenes have been found as major constituents, besides β-caryophyllene, for example sabinene in P. spruceanum [24,25], myrcene in P. heptaphyllum [15], limonene in P. lewelynii and p-cymenene in P. hebetatum [2]. Spathulenol (19.3%) and β-caryophyllene oxide (14.1%), the main constituents found in the essential oil investigated from leaves of P. confusum, have not been reported before in such high percentages in other Protium spp.

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Concerning the essential oil from fruits, our sample, mainly constituted of limonene, 3-carene and p-cymene, is rather different from those reported earlier for other species of Protium. α-Terpinene [1] and α- and β-pinene [15] were described as the major components of the oil from fruits of P. heptaphyllum, whereas terpinene derivatives were found in P. icicariba [23], and sabinene and limonene in P. spruceanum [25]. However, very little data on the volatile constituents from bark and stems of Protium spp. are available in the literature reviewed. Sabinene and limonene have been reported as major components of the bark oil of P. spruceanum [25], and terpinolene has been found in a high percentage in the oil from stems of P. heptaphyllum [22]. Both samples show a different composition from the essential oils of P. confusum. Results on the antimicrobial and larvicidal activities are shown in Table 2. Three out of the four essential oils investigated exhibited antibacterial activity against Staphylococcus aureus and Mycobacterium smegmatis. For both microorganisms, the MIC values in the case of the oil from leaves, which was the most active, were found to be 62.5 μg/mL. This activity could be in part due to the high β-caryophyllene oxide content of the sample, a compound for which antibacterial activity, particularly against S. aureus, has been previously reported [26,27]. No activity was detected against the other microorganisms tested. In contrast, the bark oil showed the highest larvicidal activity against A. aegypti with a LC100 value of 125 μg/mL, followed by the oils from leaves, stems and fruits with LC100 values of 250, 500 and 500 μg/mL, respectively. In conclusion, the essential oils from different plant parts of P. confusum have an original chemical composition rather different from those reported earlier for other species of Protium. The major constituent of the fruit oil is limonene, whereas spathulenol, β-caryophyllene oxide, and β-caryophyllene are the characteristic main components of the leaf oil. The bark and stem essential oils have p-cymen-8-ol, spathulenol, and hexadecanoic acid as major constituents. Furthermore, the oils from bark, stems, and especially leaves are active against Staphylococcus aureus and Mycobacterium smegmatis. Additionally, the bark oil has a strong larvicidal activity against Aedes aegypti.

Table 2: Antimicrobial (MIC) and larvicidal (LC) activities of the essential oils from different parts of Protium confusum.

MICa,b LCa

Essential oils and reference substance St

aphy

loco

ccus

aur

eus

Myc

obac

teri

um

smeg

mat

is

Can

dida

alb

ican

s

Aede

s aeg

ypti

Leaves 62.5 62.5 n.a. 250 Fruits n.a. n.a. n.a. 500 Bark 500 500 n.a. 125 Stems 500 500 n.a. 500 Streptomycin sulphate 12.5 12.5 - - Amphotericin B - - 0.391 - Tetramethrin - - - 0.4

a Values in µg/mL. n.a.: not active (> 1000 μg/mL). -: not tested. b Activity was also tested against Escherichia coli, Salmonella gallinarum, Klebsiella pneumoniae, and Pseudomonas aeruginosa, but none of the four oils was active against them.

Experimental

Plant material: Leaves, stems, bark and fruits from Protium confusum (Rose) Pittier were collected in Llano Cartí (Rep. Panama). Plant material was authenticated by Prof. Mireya Correa (Director of the Herbarium of the Universidad de Panamá) and a voucher specimen was deposited in the Herbarium of the Universidad de Panamá under the number FLORPAN 2932 (PMA). Essential oil isolation: Air-dried leaves, fruits, stems and bark were submitted separately to hydrodistillation for 3 h using the Clevenger type apparatus described in the European Pharmacopoeia [28]. GC and GC-MS analysis: Analyses of the volatile oils were carried out by GC-FID and GC-MS using two fused silica WCOT capillary columns with different stationary phases: Supelcowax™ 10 and methylsilicone SE-30 columns (30 m x 0.20 mm, 0.25 µm film thickness). GC-FID analyses were performed on a Hewlett-Packard 6890 instrument, using the following analytical conditions: carrier gas, helium; flow rate, 1 mL min-1; oven temperature programmed from 80 to 220ºC at a rate of 4ºC min-1; injector temp., 250ºC; detector temp., 270ºC; split ratio, 1/60. Mass spectra were obtained with a computerized system constituted of a GC Hewlett-Packard 5890 series II coupled to a mass selective detector Hewlett-Packard 5971A, using the same analytical conditions as above. Mass spectra were taken over m/z 35-400, using an ionizing voltage of 70 eV.

Essential oils of Protium confusum Natural Product Communications Vol. 4 (10) 2009 1405

Identification and quantification: Essential oil constituents were identified on the basis of their GC retention indices, determined with reference to a homologous series of fatty acid methyl esters, and by comparison of fragmentation patterns of their mass spectra with those stored in our own library and in the GC-MS database, and with literature data [29]. Quantification of each compound was performed on the basis of its uncorrected GC peak areas in both columns. Antimicrobial activity: The antimicrobial activity of the essential oils, dissolved in DMSO at a concentration of 1000 μg/mL, was assayed against two Gram-positive bacteria Staphylococcus aureus (ATCC 6538) and Mycobacterium smegmatis (ATCC 607), four Gram-negative bacteria Escherichia coli (ATCC 9637), Salmonella gallinarum (ATCC 9184), Klebsiella pneumoniae (ATCC 10031), and Pseudomonas aeruginosa (ATCC 27853), as well as against the yeast Candida albicans (ATCC 10231), following the method described by [30]. The standard antibiotics streptomycin sulfate and amphotericin B were used as positive controls. The bacteriostatic and fungistatic activities were determined by measuring the minimum inhibitory concentration (MIC) from diluted samples of 500, 250, 125, 62.5, 31.25, 15.6

and 7.8 μg/mL. All the experiments were performed in triplicate and the results expressed as mean values. Larvicidal activity: The larvicidal activity was tested against Aedes aegypti, according to the method used by Cepleanu [31], with some modifications [32]. Briefly, eggs of A. aegypti, provided by the Ministry of Health of Panama, were incubated at 29-32oC for 24 h in tap water, previously stored for 72 h, after which larvae were between stages I and II of development. Five to eight mg of essential oil was dissolved in 50 μg of DMSO and the concentration was adjusted to 1000 μg/mL with stored tap water. Essential oil concentrations of 500, 250, 125, 62.5, 31.2, 15.6 and 7.8 μg/mL were assayed in 96% microwell titer plates. In each well, 100 μL of a suspension containing 10-15 larvae were added and incubated at 25-28oC for 24 h in the dark. The minimum concentration at which all larvae died (LC100) was determined using a binocular microscope 10x. Tetramethrin, with a LC100 value of 0.4 ± 0.02 μg/ml, was used as positive control. Acknowledgments - Authors are grateful to the SENACYT (Rep. Panama, project # 11-2004) and to the Organization of American States (project AE 036/07) for financial support.

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