0MALARIA JOURNAL
RESEARCH
Open Access
Anti-malarial activity and HS-SPME-GC-MS chemical profiling of Plinia cerrocampanensis leaf essential oil
Armando A Durant1,3*1, Candelario Rodríguez1, Liuris Herrera2, Alejandro Almanza2, Ana I Santana
Carmenza Spadadora2+ and Mahabir P Gupta'
Abstract
Background: Plinia cerrocampanensis is an endemic plant of Panama. The leaf essential oil of this plant has shown antibacterial activity. However, anti-malarial activity and chemical profiling by HS-SPME-GC-MS of this essential oil have not been reported before.
Methods: Anti-malarial activity of the essential oil (EO) was evaluated in vitro against chloroquine-sensitive HB3 and chloroquine-resistant W2 strains of Plasmodium falciparum. Synergistic effect of chloroquine and the EO on parasite growth was evaluated by calculating the combination index. A methodology involving headspace solid phase microextraction and gas chromatography-mass spectrometry (HS-SPME-GC-MS) was developed to investigate the composition of Plinia cerrocampanensis EO.
Results: Plinia cerrocampanensis EO showed a high anti-malarial activity and a synergistic interaction with chloroquine. The Plinia cerrocampanensis EO inhibited P. falciparum growth in vitro at an IC50 of 7.3 |g/m_. Chloroquine together with the EO decreased the IC50 of chloroquine from 0.1 |g/m_ to 0.05 |g/m_, and of the EO from 7.3 |g/m_ to 1.1 |g/m_. The measured combination index was 0.58, which clearly indicates that the EO acts synergistically with chloroquine. Since the EO maintained its inhibitory activity on the chloroquine-sensitive strain of the parasite, it could be acting by a different mechanism of action than chloroquine. The best HS-SPME-GC-MS analytical conditions were obtained when the temperature of extraction was 49°C, incubation time 14 min, and the time of extraction 10 min. This method allowed for the identification of 53 volatile constituents in the EO, including new compounds not reported earlier.
Conclusions: The anti-malarial activity exhibited by the Plinia cerrocampanensis EO may lend support for its possible use as an alternative for anti-malarial therapy.
Keywords: Malaria, Plinia cerrocampanensis, Plasmodium falciparum, Essential oil, Anti-malarial activity, Solid phase microextraction, Gas chromatography-mass spectrometry
Background
Malaria is a life-threatening tropical disease caused by five species of Plasmodium, the most deadly of them in humans being Plasmodium falciparum. According to the World Health Organization (WHO), malaria affected
* Correspondence: adurant@indicasat.org.pa tEqualcontributors
Center for Drug Discovery and Biodiversity, Institute of Scientific Research and Technology Services (INDICASAT), Panama, Panama 3Faculty of Natural, Exact Sciences and Technology, University of Panama, Panama, Panama
Fulllist of author information is available at the end of the article
219 million people around the world in 2010, causing over 600,000 deaths. On the other hand, increased rate of resistance to drugs used for treating patients has been observed over the past 20 years [1]. Chloroquine resistance has been reported in most of the malaria-endemic regions, while resistance to artemisinin has been detected in Cambodia, Thailand, Myanmar, and Vietnam [1]. These facts highlight the challenge in discovery of new drugs and new strategies for combating the disease. Historically natural products, such as aromatic plants, have been a source of new drugs and pharmacological
O© 2014 Durant et al.; licensee BioMed Central Ltd. This is an open access article distributed under the terms of the Creative Centfäl Commons Attribution License (http://creativecommons.Org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.
alternatives for treating several diseases. The essential oils (EOs) are complex mixtures of terpenes and to a lesser extent of non-terpenoid compounds. Extensive research has been done on chemical characterization and determination of the antibacterial, antifungal, antileish-manial, and antiplasmodial activities of the EOs extracted from a wide variety of plants [2-7].
Plinia cerrocampanensis (Myrtaceae) is a tree that grows to a height of c.8 m at an altitude of 800 and 1,000 m in the surroundings of Cerro Campana National Park (Panama) [8]. The EO of this plant has been reported to possess antimicrobial activity against Staphylococcus aureus, Pseudomonas aeruginosa, Microsporum gypseum, Trichophyton mentagro-phytes, Trichophyton rubrum, and Helicobacter pylori [9]. However, its anti-malarial activity has not been reported before. Some terpenes have shown chemo-therapeutic activity. For the extraction of the different terpenoids, and other compounds that constitute an EO, head-space solid phase microextraction (HS-SPME) is a powerful tool that requires a minimal amount of sample. The isolated compounds are separated and identified by using GC-MS. SPME, which is a simple, fast, sensitive, solvent-free method developed by Pawliszyn et al. [10,11] in the early 1990s, and used for research in many fields, such as cancer [12], medicinal plants [13] and metabolomic analysis [14]. This methodology was used by Da Porto and Decorti [15] for the determination of the aroma profile of the EO obtained by hydro-distillation of lavender (Lavandula angustifolia) flowers.
The present study aimed to (a) evaluate in vitro anti-malarial activity of Plinia cerrocampanensis EO against P. falciparum; (b) study the possible synergistic inhibitory effect of chloroquine and the EO on the growth of the parasite; and (c) optimize a new HS-SPME-GC-MS methodology for identification of the volatile constituents of Plinia cerrocampanensis EO, which could possibly explain the observed anti-malarial activity.
Methods
Plant material and extraction of the essential oil
Fresh leaves of Plinia cerrocampanensis were collected in the Altos de Campana National Park (Province of Panama, Panama). The plant was identified by Alex Espinosa, taxonomist of CIFLORPAN, University of Panama. Leaves were protected from humidity and light until extraction of the EO. A voucher specimen No. 6623 is deposited in the Herbarium of the University of Panama (PMA). Plinia cerrocampanensis EO was extracted from 100 g of the collected plant material by hydrodistillation, for three hours, using a Clevenger-type apparatus as described in the European Pharmacopeia [16].
Cytotoxicity assay
Vero cells were seeded in 96-well plates using RPMI 1640 medium (Sigma-Aldrich, USA) supplemented with 10% foetal bovine serum (Gibco, Invitrogen, USA) and a combination of 1% penicillin/streptomycin. The cells were allowed to grow for 24 hours before adding the EO, dissolved in dimethyl sulphoxide (DMSO), to final concentrations of 10, 4, 0.2 and 0.08 ^g/mL (w/v). A negative control (with an equal volume of DMSO) was placed in all plates and counted as 100% growth. All samples were incubated for five days before staining and examining for reduction of 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) (Sigma Aldrich, USA) and analysed four hours later in an ELISA plate reader at a wavelength of 570 nm [17].
Anti-malarial activity
The W2 and the HB3 strains of P. falciparum were obtained from the Malaria Research and Reference Reagent Resource Centre (MR4, Manassas, VA, USA), and maintained in continuous culture for about two months before new stocks were thawed. The parasites were grown and maintained in cultures using the method of Haynes et al. [18]. All chemicals except sodium bicarbonate and genta-micin (Gibco; Invitrogen, USA), were purchased from Sigma-Aldrich (USA). Cultures consisted of a 2% haemato-crit suspension of O+ human erythrocytes in RPMI 1640 medium supplemented with a gentamicin solution at 0.01 mg/mL, 25 mM HEPES buffer, 25 mM NaHCO3, and 10% human serum. Cultures were fed with a gas mixture consisting of 5% CO2, 5% O2, 90% N2 and incubated in a cyclic incubator to achieve synchronization by temperatures, as described by Almanza et al. [19]. Estimation of the parasitaemia as well as visualization of parasite prior to the assays were done using normal light (Giemsa stain) microscopes. All samples were run in duplicate. The assay was carried out in 96-well plates at final concentrations of 10, 2, 0.4 and 0.08 ^g/mL and re-evaluated at higher or lower concentrations as necessary. The final dilution contained less than 1% DMSO, which had no measurable effect on parasite survival. DMSO at a final concentration of 1% in RPMI 1640 culture media was used as a negative control. The positive control consisted of chloroquine (CQ) at concentrations of 1,000, 200, 100, 50, and 10 nM. The parasites were incubated for 48 hours at which point 50 ^L of a PicoGreen cocktail (Invitrogen, USA) were added to each well and the fluorescence determined after 30 min at 485 nm in a fluorometer. To measure the effect of the samples alone on the fluorescence signal, each extract concentration was incubated in the absence of parasites and the signal was subtracted from the value obtained in the presence of samples and parasites. For the synergistic studies, different doses of CQ (10, 50, 100, 200 and 1,000 nM) and Plinia cerrocampanensis EO (0.08, 0.4, 2 and10 ^g/ml) were
used, maintaining constant the concentration of one while varying the other.
Statistical analysis was carried out with the LSW DATb add-on of Microsoft Office Excel. The significance level was p <0.05.
Solid phase microextraction fibre selection
Three fibres were evaluated in this research: car-boxen-polydimethylsiloxane StableFlex (CAR/PDMS, 75 ^m), polydimethylsiloxane-divinylbenzene Stable-Flex (PDMS/DVB, 65 ^m), and divinylbenzene-carboxen-polydimethylsiloxane StableFlex (DVB/CAR/ PDMS 50/30 ^m) (Supelco, Bellefonte, PA, USA). All fibres were conditioned in the gas chromatograph injector port at the temperature and time recommended by the manufacturer.
Five hundred milligrams of Plinia cerrocampanensis EO (liquid matrix) were transferred to a 10 mL vial and hermetically sealed. The sample was equilibrated during 20 min at 30°C in a thermostatic bath. Then the fibre was exposed to sample headspace for 15 min. After the sampling period, the SPME fibre was inserted into the GC injector port and maintained there for 7 min at 250°C for desorption of compounds.
Experimental design
The times of incubation and extraction and temperature of extraction are critical parameters that determine the equilibrium, vapour pressures and extraction efficiency of volatiles in the headspace [20]. These variables, which strongly influence the efficiency of the HS-SPME-GC-MS, were optimized by using a Doehlert design. Sixteen randomized experiments were performed in duplicate. The total GC-MS peak areas were used as response. Statistical analysis was done using Statgraphics Plus 5.1 (Statistical Graphics, Rockville, MD, USA).
Gas chromatography-mass spectrometry analysis
All the analyses were performed on an Agilent 6890 N gas chromatograph (GC) equipped with a 5975C mass-selective detector (MSD) with triple-axis detector (Agilent Technologies, Palo Alto, CA, USA). The separation was achieved on a DB5-MS capillary column (30 m length, 0.25 mm id, 0.25 ^m film thickness), Agilent Technologies, Palo Alto, CA, USA).
Gas chromatograph oven initial temperature was kept at 50°C for 2 min, then increased to 240°C at 6°C min-1 and held for 5 min. The injector temperature was maintained at 250°C, using splitless injection mode (2 min). Helium was used as carrier gas with a constant flow-rate of 1 mL min- . The mass spectrometer was operated in scan mode from 30-550 m/z; ion source temperature was set at 250°C; the ionization was performed in the impact ionization mode (EI) with the ionization voltage set to 70 eV.
Identification of the compounds was carried out based on comparison of their fragmentation patterns using authentic standards when available and the NIST 11 Mass Spectral Search Program (NIST, Washington, USA), and with data from the literature [21]. Further identification of the compounds was achieved by comparison of their calculated experimental linear Kovat's retention indices (KI) with those reported in the literature using chromatographic columns of similar stationary phase composition.
Results
Headspace solid phase microextraction fibre selection
The first HS-SPME parameter optimized for characterization of the metabolites of Plinia cerrocampanensis EO was the selection of the SPME fibre. In this study three
Table 1 MS detector peak area of some volatile metabolites in Plinia cerrocampanensis essential oil analysed by using three different SPME fibres
Mean MS response value (x107)
Compound DVB/CAR/PDMS CAR/PDMS DVB/PDMS
1 Furfural 67.2 - -
2 a-Pinene 584.0 632.0 894.0
3 Benzaldehyde 257.0 - 201.0
4 y-Terpinene 1150.0 799.5 -
5 Terpinolene 547.0 - -
6 Linalool 2390.0 - 2630.0
7 Neo-allo-ocimene 106.0 239.7 30.8
8 4-Terpineol 599.0 467.0 660.0
9 a-Terpineol 231.0 194.0 261.0
10 Nerol 12.9 8.8 15.5
11 Geraniol 26.8 14.3 30.8
12 a-Copaene 663.0 639.0 741.0
13 Sativene 11.1 - -
14 a-Cedrene 140.0 - 229.0
15 Caryophyllene 114.0 112.0 132.0
16 a-Bergamotene 112.0 105.1 129.0
17 Epi-ß-santalene 33.0 - -
18 a-Humelene 46.1 46.7 55.5
19 ß-Acoradiene 69.2 81.3 93.2
20 Y -Muurolene 89.6 87.2 103.0
21 a-Muurolene 80.3 53.5 96.4
22 a-Curcumene 191.0 170.9 184.0
23 ß -Bisabolene 907.0 480.3 1100.0
24 5-Cadinene 291.0 277.0 328.0
25 frons-Nerolidol 195.0 83.8 243.0
26 Cubenol 11.8 8.4 -
27 Bisabololoxide II 37.9 - 47.7
28 a-Bisabolol 159.0 58.0 213.0
Table 2 Experimental Doehlert design matrix used for optimization of extraction parameters of Plinia cerrocampanensis essential oil by HS-SPME
Experiment Extraction temperature (°C) Time of equilibrium (min) Time of extraction (min) Total peak area (109)
1 45 25 20 136.86
2 60 25 20 1.41
3 52 25 35 6.84
4 52 40 25 9.47
5 30 25 20 50.99
6 38 25 5 43.98
7 38 10 15 111.39
8 52 25 5 139.92
9 52 10 15 137.97
10 38 25 35 106.08
11 45 10 30 100.77
12 38 40 25 100.85
13 45 40 10 122.17
14 45 40 10 121.96
15 45 40 10 122.20
16 45 40 10 120.34
commercially available SPME fibres, with two or three different coating polymers, were compared for assessing their concentration capacity. SPME fibres with more than one coating have shown to be the most effective when studying medicinal and aromatic plants [22].
The extraction efficiency of each fibre was determined on the basis of two main criteria, the total MS-detector peak area and the number of identified compounds. The DVB/ CAR/PDMS fiber permitted the identification of a larger number of constituents of the EO than CAR/PDMS and DVB/PDMS fibers. Furthermore, DVB/CAR/PDMS total peak response (9.12 x 1010) was higher than that obtained with DVB/PDMS (8.42 x1010) and CAR/PDMS (4.55 x 1010) fibres (Table 1). DVB/CAR/PDMS is an adsorption-
based fibre made of polydimethylsiloxane (PDMS) coating that allows the absorption of less polar substances; car-boxen (CAR) and divinylbenzene (DVB) porous coating materials that provide high selectivity and sensitivity for more polar compounds. This fibre was selected as the most appropriate for obtaining the widest composition profile of Plinia cerrocampanensis EO, and was used in all further experiments.
Optimization of the HS-SPME extraction procedure
The times of extraction and incubation and temperature of extraction are important factors that determine the efficiency of the overall HS-SPME extraction process [23]. Thus, these parameters were optimized by using a three-
Standardized Effect
Figure 1 Pareto chart of standardized effects of extraction temperature, time of incubation and time of extraction.
variable Doehlert design. This multivariate method allows for the simultaneous evaluation of the factors under study. Table 2 shows the experimental Doehlert design matrix and the response in terms of total area of the compounds identified from Plinia cerrocampanensis EO (liquid matrix). The design consisted of 16 randomized experiments to avoid systematic errors. Analysis of variance (ANOVA) was performed for determining the statistical significance (p <0.05).
Figure 1 shows the Pareto chart of the standardized effects. As can be observed, all three of the evaluated factors were significant, and increasing their value affected negatively the extraction. Among the HS-SPME parameters studied the time of extraction had the greatest influence on the extraction efficiency, while the temperature of extraction was less significant.
HS-SPME is an equilibrium process among three phases, the sample matrix, a gaseous headspace and the fibre coating. A first equilibrium is established between the analytes in the matrix and the headspace; subsequently, the SPME fibre is introduced in the headspace leading to a new equilibrium between the analytes in the headspace and in the fibre [24]. By raising the temperature the amount of analytes released from Plinia cerrocampanensis EO and extracted by the SPME fibre increased until an optimal value at 49°C (Figure 2A), after which higher temperatures influenced negatively the results. As the extraction temperature increases the Henry's constant of the analytes also increases, as well as the amount of compounds released from the liquid phase, especially less volatile compounds. Both physi-cochemical phenomena improve the extraction efficiency. Nevertheless, above 49°C the amount of compounds extracted by the SPMEdvb/car/pdms fibre tends to decrease due to desorption of compounds from the fibre.
Incubation time influences the number of compounds that are transferred from the liquid matrix and reach the headspace. The effect of this variable on analytical response is shown in Figure 2. A maximum response was observed at 14 min. No improvement on extraction of EO metabolites was observed by increasing the incubation time.
In order to assess the effect of extraction time on the analysis, the SPMEdvb/car/pdms fibre was introduced into the sample headspace for different periods. The optimal response was achieved with an extraction time of 10 min (Figure 2). HS-SPME is an equilibrium process influenced strongly by the partition coefficient of the compounds between the liquid phase and the headspace
(Kfiber/matrix)'
Withdrawing the SPME fibre from the headspace before 10 min did not allow this equilibrium to be achieved, and hence less metabolites were extracted. conversely, higher time of extraction reduced the total volatile recovery due to diffusion of the analytes from the fibre [25].
30 35 40 « 50 55 ^ 0 Temperature of Extraction
Time of Extraction
30 35 « 45 50 55 Temceraiure of Extraction
Incubation Time
Time of Extraction
20 25 30 Jy 4U ■ incubation Time
Figure 2 Fitted response surface plot. (A) Extraction temperature (°C) vs time of extraction (min). (B) Extraction temperature (°C) vs time of incubation (min). (C) Time of incubation (min) vs time of extraction (min).
According to the experimental results, the best analytical response was obtained when the temperature of extraction was 49°C, incubation time 14 min, and time of extraction 10 min. These optimal values were selected for extraction of the EO metabolites in all further experiments.
Chemical composition of the essential oil of Plinia cerrocampanensis by HS-SPME-GC-MS
This is the first time that the chemical composition profiling of Plinia cerrocampanensis EO has been studied by
Table 3 Chemical composition of Plinia cerrocampanensis B. essential oil by HS-SPME-GC-MS
Peak no Compound KIa m/z Perce
1 Furfural 830 95-67-42 0.21
2 a-Pinene 933 93-79-41 1.96
3 Benzaldehyde 958 105-77-51 1.10
4 Myrcene 982 41-93-69 0.91
5 3-5-Carene 1006 93-79-41 0.90
6 o-Cymene 1020 119-91-134 6.85
7 frons-p-Ocimene 1041 93-79-41 2.62
8 c/s-p-Ocimene 1053 93-91 -79 2.58
9 y-Terpinene 1067 93-77-41 4.70
10 Terpinolene 1092 93-121-136 2.01
11 Linalool 1104 43-71 -55 10.47
12 Fenchol 1121 81 -43-67 0.06
13 c/s-Sabinol 1133 92-81-109 0.11
14 Neo-allo-ocimene 1140 121-105-79 2.72
15 1,4-Dimethyl-4-acetyl-1-cyclohexene 1157 109-67-43 0.04
16 Trons,cis-2,6-Nonadienal 1163 41 -70-39 0.05
17 Terpin-4-en-1-ol 1174 43-71 -93 2.22
18 m-Methylacetophenone 1188 119-91-65 0.15
19 a-Terpinol 1196 59-43-93 1.20
20 P-Cyclocitral 1214 41-137-123 0.06
21 Nerol 1229 41 -69-81 0.12
22 p-Menth-1(7)-en-2-one 1241 82-81-152 0.07
23 Carvenone 1248 95-110-67 0.18
24 Geraniol 1255 41 -69-81 0.29
25 c/s-2-Decenal 1272 41 -55-70 0.04
26 Carvacrol 1313 135-91-150 0.05
27 a-Copaene 1365 105-119-161 8.86
28 Sativene 1390 108-41-91 0.19
29 Ylangene 1400 105-119-93 0.89
30 a-Cedrene 1412 119-41-105 0.92
31 frons-p-Caryophyllene 1420 41-91-79 1.34
32 a-Bergamotene 1440 41-93-119 1.93
33 Y-Elemene 1449 121-41-93 1.24
34 Epi-p-santalene 1453 94-41-79 0.52
35 a-Humelene 1458 93-41-67 0.54
36 p-Farnesene 1462 41 -69-91 1.97
37 P-Acoradiene 1469 119-41-105 1.05
38 Y-Gurjunene 1476 105-91-81 0.82
39 Y -Muurolene 1484 161-41-105 1.42
40 a-Curcumene 1490 119-132-41 3.83
41 Indipone 1499 149-93-41 1.18
42 a-Muurolene 1507 105-41-161 0.95
43 p-Bisabolene 1513 41 -69-93 16.26
44 5-Cadinene 1527 119-161-41 4.21
Percentage composition (%)
Table 3 Chemical composition of Plinia cerrocampanensis B. essential oil by HS-SPME-GC-MS (Continued)
45 y-Bisabolene 1540 41-93-107 2.19
46 trans- Nerolidol 1558 41-69-93 2.68
47 Spathulenol 1569 43-91 -79 0.27
48 tau-Cadinol 1632 43-161-81 0.26
49 Cubenol 1649 43-161-95 0.26
50 Bisabololoxide B 1666 43-59-105 0.92
51 P-Bisabolol 1674 41 -67-93 0.15
52 a-Bisabolol 1687 43-119-109 3.46
53 a-Farnesol 1709 41 -69-81 0.01
aRetention index. Components are listed in increasing order of their retention indices on DB5-MS.
using HS-SPME-GC-MS approach. Table 3 lists the constituents identified in the EO analysed. Fifty-three compounds were identified by this methodology. The main constituents (more than 2% of the identified compounds) were fi-bisabolene, fi-linalool, a-copaene, o-cymene, y- terpinene, 5-cadinene, a-curcumene, a-bisabolol, neo-allo-ocimene, trans-nerolidol, trans-fi-oci-mene, fi-ocimene, y-bisabolene and terpinolene (Table 3). Vila et al. have reported the chemical composition of the EO obtained from leaves of Plinia cerrocampanensis utilizing two different GC-columns, i e, a non-polar and a polar phase columns, identifying 40 compounds: 33 compounds were identified on the non-polar phase column, and 31 on the polar phase column [9]. Clearly, the more sensitive HS-SPME-GC-MS methodology developed in this study allowed the identification of a higher number of compounds in a single chromatographic run.
The current investigation reports for the first time new compounds present in the EO obtained from leaves of Plinia cerrocampanensis, such as neo-allo-ocimene, y-elemene, caryophyllene, acoradiene, y-muurolene, and fi-myrcene.
Sesquiterpene hydrocarbons (49.13%), and monoter-pene hydrocarbons (25.25%) were the most abundant chemical class of compounds identified by HS-SPME-GC-MS, followed by oxygenated monoterpenes (15.03%), oxygenated sesquiterpenes (9.19%) and aldehydes (1.35%).
Anti-malarial activity
This is the first report on the anti-malarial activity of the EO from Plinia cerrocampanensis. The antiplasmodial effects of Plinia cerrocampanensis EO were determined by quantifying the growth inhibition of the CQ-resistant P. falciparum W2 strain when compared with parasites cultured in a medium free of the EO. The Plinia cerro-campanensis EO inhibited parasite growth in vitro at IC50 7.3 ^g/mL. This value indicates a high antiplasmo-dial activity. The activity of the oil was lower than that of chloroquine (IC50 = 0.1 ^g/mL), which could result
from several factors, one of them being the dilution of the active compound (s) in the matrix.
To get an idea of the mechanism by which the EO was acting on P. falciparum, its activity against a CQ-sensitive strain, HB3, was tested and compared the results with those obtained with the CQ-resistant W2. While CQ shows difference in IC50 in the resistant (IC50 = 0.1 ^g/mL) and sensitive (IC50 = 0.01 ^g/mL) strains, the EO maintained its level of activity, in both the resistant (IC50 = 7.3 ^g/mL) and sensitive (IC50 = 10.2 ^g/mL) strains. These results suggest that the EO may be using another mechanism that does not involve inhibition of fi-haematin formation nor inhibition of the peroxidative degradation of haemin, since CQ and other quinolones with anti-malarial activity are thought to act through these two mechanisms [26,27].
The EO caused toxicity to mammalian cells at a concentration of 28.6 ^g/mL, which is four-fold higher than the concentration at which the oil showed antiplasmo-dial effects, projecting a reasonable therapeutic window for its use in vivo. The HS-SPME-GC-MS methodology developed in this study allowed the identification of ter-penes and terpenoids in the EO of Plinia cerrocampa-nensis: a-pinene, fi-linalool, trans-nerolidol, a-bisabolol and a-farnesol, which have been reported to possess antiplasmodial activity and, therefore, may explain the observed anti-malarial activity of EO [28-30].
The monoterpene a-pinene displayed a stronger anti-malarial activity than quinine when tested against a CQ-resistant strain of P. falciparum, mainly affecting the ring stage of the parasite [29]. fi-linalool and farnesol arrest the growth of the apicomplexan mainly by (a) affecting the dolichol biosynthesis at the trophozoite and schizont stages; and, (b) inhibiting the biosynthesis of the isoprene side chain of the benzoquinone ring of ubi-quinones [30]. Treatment of P. falciparum with the isolated oxygenated sesquiterpene a-bisabolol caused inhibition in the development of parasites [30]. Macedo et al. demonstrated the high activity of the sesquiterpene trans-nerolidol against P. falciparum by decreasing its ability to biosynthesize the isoprene chain attached to
° 100
\(EC 50% contour line
......
0 2 4 6
EOng/ml
Figure 3 Isobologram and combination index (CI) of chloroquine (CQ) and the essential oil (EO) of Plinia cerrocampanensis. The 50% contour line shows a less than 1 combination index which shows synergy between EO and CQ at concentration (EO, CQ).
coenzyme Q [31]. In most cases, the anti-malarial activity displayed by the Plinia cerrocampanensis EO was higher than the activity observed for these compounds, suggesting that trace components of the oil could be enhancing the effect of the active components. A possible synergism among the different constituents of the oil could also be responsible for its anti-malarial activity.
To determine possible synergistic or additive effects when applied with other drugs, the activity of the EO was examined in conjunction with CQ at different concentrations. Synergism or additive effect was evaluated by calculating the combination index (CI) as described by Chou and Talahay [32] using the following formula:
(D)x , (D)2
(Dx)i (Dx)2 (DRI)i (DRI)2
where D is the concentration at which both compounds show the same desired efficacy (ED) in combination, and Dx is the concentration at which each of the compounds alone show this ED [32]. A synergistic effect is achieved if CI is <1.0 while an additive effect is obtained at CI = 1.0. The application of CQ together with the EO on strains of P. falciparum, decreased the IC50 for CQ from 0.1 ^g/mL to 0.05 ^g/mL, and for the EO from 7.3 ^g/mL to 1.1 ^g/mL (Figure 3).
This means that a higher in vitro activity against the parasite was obtained for the oil and CQ when applied together. The measured CI combination was 0.58, which clearly indicates that the EO acted synergistically with CQ. Moreover, the therapeutic window of the EO increased substantially in the presence of a very small amount of CQ. Based on the physico-chemical properties of the EO, it could also cause the disruption of the parasite cell membrane, which affects the permeability of the cell and the transfer of important biochemical
substances needed by the parasite for sustaining crucial metabolic processes. The reduction in the concentration of CQ required for achieving its therapeutical effect may not only lower the incidence of adverse reactions in humans, but most importantly, may promote the use of this cost effective drug in the large regions worldwide where resistance to it has been observed.
Conclusions
This research demonstrated that the EO of Plinia cerrocampanensis could be a potential alternative for the treatment of malaria. Moreover, due to the synergistic anti-malarial activity of the EO and the CQ, this could be used in combination with CQ as anti-malarial therapy. The studies are underway to determine the anti-malarial effect of major isolated compounds in the EO of Plinia cerrocampanensis.
The developed HS-SPME-GC-MS methodology allowed for the identification of new constituents in the EO of Plinia cerrocampanensis. Monoterpenes and sesquiter-penes, which constitute most of the volatile organic compounds of the essential oil, could be responsible for the bioactivity showed by the oil in this study.
Abbreviations
EO: Essentialoil; HS-SPME: Headspace-solid phase microextraction; GC: Gas chromatography; MS: Mass spectrometry.
Competing interests
The authors declare that they have no competing interests. Authors' contributions
CS, MPG and AAD participated in research design and criticalreview of the manuscript; MPG supplied plant materialand suggested this research; AIS, CR, LH, AA and AAD conducted experiments; AIS, CS and AAD performed data analysis; CS and MPG contributed in the writing and revision of the manuscript; AAD drafted and wrote the finalmanuscript. Allauthors approved the finalmanuscript.
Acknowledgements
AD, CS and MPG would like to acknowledge SENACYT, Panama for supporting the NationalSystem of Investigators (SNI) and the Organization of American States for support to CIFLORPAN.
Author details
Center for Drug Discovery and Biodiversity, Institute of Scientific Research and Technology Services (INDICASAT), Panama, Panama. 2Center for Cellular and Molecular Biology of Diseases, Institute of Scientific Research and Technology Services (INDICASAT), Panama, Panama. 3Faculty of Natural, Exact Sciences and Technology, University of Panama, Panama, Panama. 4Center of Pharmacognostic Research on Panamanian Flora (CIFLORPAN), College of Pharmacy, University of Panama, P.O. Box 0824-00172, Panama, Panama.
Received: 15 August 2013 Accepted: 9 January 2014 Published: 13 January 2014
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doi:10.1186/1475-2875-13-18
Cite this article as: Durant et al.: Anti-malarial activity and HS-SPME-GC-MS chemical profiling of Plinia cerrocampanensis leaf essential oil.
Malaria Journal 2014 13:18.
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