Essential Oil of Mitracarpus Frigidus as a Potent Source of Bioactive Compounds

In our previous work (Fabri et al. 2009), we showed that different extracts of Mitracarpus frigidus had significant antibacterial, antifungal and leishmanicidal activities. In order to increase our knowledge about this species, this work assesses the chemical composition and the in vitro biological activity of its essential oil. Thus, the essential oil obtained by hydrodistillation of the aerial parts of M. frigidus was analyzed by GC/MS. Among several compounds detected, 11 were identified, being linalool and eugenol acetate the major components. The essential oil exhibited a moderate antibacterial effect against Staphyloccocus aureus, Bacillus cereus, Pseudomonas aeruginosa and Enterobacter cloacae (MIC 250 μg/mL). On the other hand, it showed a strong antifungal effect against Cryptoccocus neoformans (MIC 8 μg/mL) and Candida albicans (MIC 63 μg/mL). Expressive activity against L. major and L. amazonensis promastigote forms with IC 50 values of 47.2 and 89.7 μg/mL, respectively, were also observed. In addition, the antioxidant activity was investigated through DPPH radical-scavenging and showed a significative activity with IC 50 of 38 μg/mL. The cytotoxicity against Artemia salina was moderate with LC 50 of 88 μg/mL. The results presented here are the first report on the chemical composition and biological properties of M. frigidus essential oil.


INTRODUCTION
Some Mitracarpus species have ethnopharmacological importance as they are used in folk medicine for various purposes.For example, M. scaber is widely employed in traditional medicine in West Africa for headaches, toothache, amenorrhea, dyspepsia, hepatic diseases and leprosy.Among those folkloric uses, the juice of the plant is applied topically for the treatment of skin diseases (Dalziel 1936, Kerharo andAdam 1974).
The potential antimicrobial activity of the aerial parts extracts of some Mitracarpus species against bacterial and mould strains, like Staphylococcus aureus, Bacillus cereus, Pseudomonas aeruginosa, Candida albicans and Cryptococcus neoformans were already reported (Irob and Daramola 1993, Anais da Academia Brasileira de Ciências ( 2012) 84(4): 1073-1080 (Annals of the Brazilian Academy of Sciences) Printed version ISSN 0001-3765 / Online version ISSN 1678-2690 www.scielo.br/aabcRODRIGO L. FABRI et al. 1994, Sanago et al. 1996).Also, in our previous work, we showed that M. frigidus extracts had a significant antibacterial, antifungal and leishmanicidal activities (Fabri et al. 2009).So, in order to increase our knowledge about M. frigidus, this work deals with the chemical composition and the in vitro activity against Gram-positive and Gram-negative bacteria and yeasts strains, promastigote forms of Leishmania species and cytotoxicity against Artemia salina of the essential oil of this specie.Its in vitro antioxidant activity was also determined.

PLANT MATERIAL
M. frigidus aerial parts were collected in Juiz de Fora, Minas Gerais, Brazil, in May, 2006.Dr. Tatiana Konno identified the plant.A voucher specimen (CESJ 46076) was deposited at the Herbarium Leopoldo Krieger of the Universidade Federal de Juiz de Fora.

HYDRODISTILLATION OF VOLATILE OILS
Essential oil was obtained by means of hydrodistillation (3 h) of the dried plant material using a Clevenger type apparatus.Oil was dried and stored at 4°C until tested and analyzed.
Fifty microliters from the 20 mg/mL solutions were transferred to a 2 mL glass vial and the solvent was removed under vacuum (speedVac ® , SC250 model, ThermoSavant, U.S.A) for 18 h, 30°C and 10 millibar.The vial was closed with a cap sealed Teflon coated septum (Supelco, U.S.A) and placed in a heat block adjusted to 90°C.A SPME fiber (PDMS/DVB TM 65 µm, SUPELCO, U.S.A) was inserted with a manual holder through the septum and left in the headspace for 30 min.Immediately after that, the holder with the saturated fiber was analyzed by means of injection on gas chromatography (GC) injection port.Before use, the fiber was preconditioned at 230°C during 30 min in the GC injector port.

GAS CHROMATOGRAPHY/MASS SPECTROMETRY ANALYSIS
Gas Chromatography/Mass Spectrometry (GC-MS) analyses were performed on a Shimadzu QP-5050A (SHIMADZU, JP) instrument, equipped with a PTE TM -5 column (30 m, 0.25 mm, 0.25 µm, Supelco, USA), using helium as the carrier gas.The following conditions were employed for all analysis: helium at 22.3 mL/min; injector temperature maintained at 230°C; the oven at 80°C during 3 minutes and then heated to 300°C at 7°C/ min, holding for 5 min at 300°C.The split valve was closed during the first minute of injection and then opened, with a 1:10 ratio.The mass detector was set to scan from 50 to 500 m/z, at a rate of 2 scans per second.Data acquisition and handling was done via CLASS 5000 Shimadzu software.

MINIMAL INHIBITORY CONCENTRATION (MIC)
The minimal inhibitory concentration (MIC) was determined by using broth microdilution techniques for bacterial and yeasts (NCCLS 2002Perez et al. 1990).MIC values were determined in RPMI 1640 (Sigma) buffered to a pH 7.0 with MOPS (Sigma) for yeasts and Mueller Hinton broth (MHB) for bacteria.Yeasts were cultured at 30°C for 48 h in SDA and bacteria were cultured overnight at 37°C for 24 h in MHA.Sample stock solutions were two-fold diluted from 5,000 to 2.5 μg/mL (final volume = 80 μL) and a final DMSO concentration ≤ 1%.Then, 100 μL of RPMI or MHB were added onto microplates.Finally, 20 μL of 10 6 CFU/mL (according to McFarland turbidity standards) of standardized yeasts and bacterial suspensions were inoculated onto microplates and the test was performed in a volume of 200 μL.Plates were incubated at 30°C for 48 h for yeasts and 37°C for 24 h for bacteria.The same tests were performed simultaneously for growth control (RPMI + yeast and MHB + bacteria) and sterility control (RPMI or MHB + essential oil).
The test was performed in duplicate.The MIC values were calculated as the highest dilution showing complete inhibition of tested strain.

DPPH RADICAL SCAVENGING ASSAY
The free radical scavenging activity of sample solutions in methanol was determined based on their ability to react with stable 1,1-diphenyl-2picrylhydrazyl (DPPH) free radicals (Govidarajan et al. 2003).The essential oil at various concentrations (7.8 to 250 μg/mL) was added to a 152 μM solution of DPPH in methanol.After incubation at 37°C for 30 min, the absorbance of each solution was determined at 517 nm.The antioxidant activity of essential oil was expressed as IC 50 (inhibitory concentration), which was defined as the concentration (in μg/mL) of sample required to inhibit the formation of DPPH radicals by 50 %.α-Tocopherol and rutin were used as positive control.
culture was suspended to yield 2 million cells/mL (L.amazonensis or L. major) or 3 million cells/ mL (L.chagasi) after Neubauer chamber counting.Controls with DMSO and without plants samples were performed.All the tests were performed in triplicates.The viability of promastigotes was assayed after a three day incubation period with addition of MTT.The reaction was stopped with HCl in isopropyl alcohol and the optical densities were evaluated in a spectrophotometer at 570 nm (Multiskan MS microplate reader, LabSystems Oy, Helsink, Finland).The results were expressed as the concentrations inhibiting parasite growth by 50% (IC 50 ) and the percentage of inhibition of parasite growth.Amphotericin B was used as the standard drug.

CYTOTOXICITY ASSAY
Brine shrimp lethality bioassay (Meyer et al. 1982) was carried out to investigate the cytotoxicity of the essential oil.Brine shrimp (Artemia salina Leach) eggs were hatched in a beaker filled with sea water under constant aeration.After 48 h, the nauplii were collected by pipette and were counted macroscopically in the stem of the pipette against a lighted background.Solutions of the essential oil were made in seawater containing 1 % DMSO, at varying concentrations (10 to 1,000 μg/mL) and incubated in triplicate vials with 10 brine shrimp larvae.After 24 h of incubation, the nauplii were examined against a lighted background, with a magnifying glass and the number of survivors in each vial were counted and noted.Both positive (thymol) and negative (sea water containing 1 % DMSO) control assays were carried out in order to verify the susceptibility of A. salina under assay conditions employed.

STATISTICAL ANALYSIS
The IC 50 for antioxidant activity was calculated by Grafit 5.The IC 50 for leishmanicidal activity and cytotoxicity were calculated by Probit analysis.
Both were expressed as mean ± standard error (SE).The inhibition percentages for leishmanicidal activity were analyzed by Graph Pad Prisma 4. Statistical differences between the treatments and the control were evaluated by ANOVA test.

ANTIMICROBIAL ACTIVITY
The essential oil of M. frigidus exhibited a moderate antibacterial effect against Staphyloccocus aureus, Bacillus cereus, Pseudomonas aeruginosa and Enterobacter cloacae (MIC 250 μg/mL) (Table II).On the other hand, the oil showed a strong antifungal effect against Cryptoccocus neoformans (MIC 8 μg/mL) and Candida albicans (MIC 63 μg/mL).It was considered that if the extracts displayed a MIC less than 100 μg/mL, the antimicrobial activity was good; from 100 to 500 μg/mL, moderate; from 500 to 1,000 μg/mL, weak; over 1,000 µg/mL the extract was considered inactive (Holetz et al. 2002).

ANTILEISHMANIAL ACTIVITY
The effect of the essential oil of M. frigidus on the viability of promastigotes of L. major, L. amazonensis and L. chagasi was tested.The essential oil showed expres sive activity against L. major and L. amazonensis promastigote forms with IC 50 values of 47.2 ± 4.0 and 89.7 ± 8.6 μg/mL, respectively.Those forms were susceptible to the essential oil   at 108 μg/mL with 97% and 86% cell inhibition, respectively (Figure 1).Interestingly, L. major presented the greatest dose-dependent relationship.On L. chagasi, the percentage of inhibition did not varied significantly between the concentrations used.The percentage of inhibition presented by reference drug Amphotericin B at 10 μg/mL was 60 %, 90% and 86% for L. major, L. amazonensis and L. chagasi promastigotes, respectively.

ANTIOXIDANT ACTIVITY
The antioxidant activity of the essential oil of M. frigidus was investigated through DDPH radicalscavenging and showed a moderate activity with IC 50 of 38 ± 8 μg/mL.The reference controls α-tocopherol and rutin presented IC 50 of 0.2 ± 0.1 and 3 ± 1.8 μg/mL, respectively.

DISCUSSION
The essential oil exhibited a moderate antibacterial and a strong antifungal effect against Cryptoccocus neoformans and Candida albicans.Also, it showed expressive activity against Leishmania major and L. amazonensis promastigote forms.
Linalool and eugenol acetate were the major constituents of the essential oil.Linalool is a monoterpene commonly found in the essential oils of some aromatic plants.It is also obtained as a by-product in the industrial synthesis of vitamin E (Ohashi et al. 1997).Its biological activity, including antioxidant, antimicrobial, antiinflammatory, anesthetic and antitumor was already reported (Ghelardini et al. 1999, Mazzanti et al. 1998, Letizia et al. 2003, Dadasoglu et al. 2011).Also, the ability to inhibit the development of the mosquito larvae of Aedes aegypt has been attributed to linalool (Gottlieb et al. 1981).Eugenol acetate is a derivative of eugenol, which is employed as antimicrobial, anti-inflammatory, anesthetic, antiseptic, antioxidant, repellent agent, and in cosmetics and condiments (Lahlou 2004).
Cryptoccocus neoformans and Candida albicans are opportunistic pathogens commonly associated with disease in immunocompromised hosts.C. neoformans causes systemic disease, and cryptococcal infection is usually acquired by inhalation of fungal cells and can be limited to the lung or disseminate to the central nervous system, causing meningoencephalitis (Rodrigues et al. 1999).

C. albicans can cause local and systemic infection
and it is found normally in the buccal and vaginal regions (Zhang and Lewis 1997).The lipophylicity of the essential oil constituents could explain their antimicrobial activity, a characteristic that allows the partition of these compounds in lipids of cell membrane and mitochondria, increasing their permeability and leading to leakage of cellular contents (Cowan 1999).According to other authors, essential oil constituents can also act on cellular proteins located in cytoplasm membranes, including the ATPases, by their accumulation in the lipid double layer and the consequent destruction of lipid-protein interaction (Ultee et al. 2002, Burt 2004).Alternatively, a direct interaction of lipophylic compounds may occur with the hydrophobic portions of proteins (Juven et al. 1994, Sikkema et al. 1995).However, due to the large number of different chemical groups present in essential oils, it is likely that its antimicrobial activity is not related to a specific mechanism of action (Carson et al. 2002, Kalemba and Kunicka 2003, Skadamis and Nychas 2001).
Leishmaniasis is a chronic disease that can assume different fatal clinical forms ranging from selfhealing cutaneous to progressive mucocutaneous infection, and potentially visceral leishmaniasis (Tripathi et al. 2007).According to the World Health Organization (2004), leishmaniasis currently threatens 350 million people around the world and it is estimated that 2 million new cases occur each year.A common feature of volatiles compounds is their hydrophobic nature.Several studies addressing the action mode of such compounds usually point to cell membranes as the primary target as the essential oils, in general, have a passive entry through the membrane, leading to an increase of membrane permeability (Bakkali et al. 2008).
Antioxidant activity possibly proceeds from the presence of phenolic compounds such as methyl salicylate in the oil.The brine shrimp lethality assay is based on the ability to kill laboratorycultured Artemia salina nauplii brine shrimp and it is considered to be one of the most useful tool for the preliminary assessment of general toxicity (Maclaughlin 1991).LC 50 values < 250 μg/mL are considered significant for plant samples and had the potential for futher investigation (Rieser et al. 1996).
The results presented here are the first report on the chemical composition and biological properties of the Mitracarpus essential oil.

Figure 1 -
Figure 1 -Effect of the essential oil from M. frigidus on growth of L. major, L. amazonensis and L. chagasi promastigote forms.Parasites were treated with 11 to 108 µg/mL essential oil.Each bar represents the mean ± standard deviation of three different experiments.
SERIAL DILUTION ASSAY FOR DETERMINATION OF THE