Chemical and Biological Investigations

Chemical and Biological Investigations
of Pilocarpus spicatus essential oils


Pilocarpus spicatus Saint-Hillarie (Rosacea) is one of the various Pilocarpus spp. called ‘jaborandi’. Nowadays, the great importance of Pilocarpus spp. is due to the extraction of the alkaloid pilocarpine from the leaves (Pinero, 2002). The Rosacea has secretory cavities ligneous and schizo-lysigenous containing essential oils (Samos and Spitzer, 1999). The leaves of Pilocarpus microphyllus Stapf ex Wardle worth and Pilocarpus pennatifolius Lem produce 0.25-0.50% of essential oil (Lorenz and Matos, 2002).

In this way, a lot of essential oils from different species of the Pilocarpus genus have been analysed and chemical substances as terpenoids, alcohols, aldehydes, hydrocarbons aliphatic and aliphatic ketones have been described (Craveiro et al., 1979; Santos et al., 1997; Andrade-Net et al., 2000, 2002; Santos et al., 2004). In addition, Chaliapin is a coumarone extracted from P. spicatus essential oil. Experiment in vitro showed that binding of Chaliapin to glycosomal glyceraldehyde-3-phosphate dehydrogenase of T. cruzi, a protozoan was solely transmitted by Rhodnius prolixus, interfered with the flagellate development, Moreover, we observed a variety of effects of P. spicatus essential oil on Rhodnius prolixus -a vector of Chagas disease -which indicates their secondary metabolites, nowadays under investigation in our laboratory, as good candidates for the study of insect physiology, vector control population and perhaps, blockage of protozoan development in triatomine hosts.
It has well established that P. spicatus essential oil collected in the state of Ceará (Brazil) displays in vitro antibacterial activity against Pseudomonas aeruginosa and Staphylococcus aureus (Santos et al., 1997).

However, the chemical composition of P. spicatus essential oil showed qualitative and quantitative variation by the influence of local environmental conditions of soil and seasonal period of collections (Taveira et al., 2003). A variety of plants has been reported to show acetyl cholinesterase (Ache) inhibitory activity and so may be relevant to the treatment of neurodegenerative disorders such as Alzheimer’s disease (AD). (Mukherjee et al., 2007). Numerous essential oils and their monoterpene constituents have been investigated for their effects on Ache, and have shown inhibitory activity. Although, several extracts of plants and essential oils already have been investigated, this is the first account in the Pilocarpus genus. The brine shrimp lethality test (BST) is a simple and efficient method used to predict compounds or extracts as cytotoxic agents and that may have anticancer activity (Meyer et al., 1982).

Although several essential oils have been tested against brine shrimp, P. spicatus essential oil cytotoxicity has not been investigated yet. In this work, studies were carried out to elucidate the chemical composition and analyses antibacterial, anticholinesterase and cytotoxicity activities in vitro of P.spicatus essential oil from Rio de Janeiro State (Brazil). MATERIALS AND METHODS Plant material Aerial parts of P. spicatus Saint-Hillarie (Rosacea) was collected in Sandy Coastal Plains (Resting de Jurubatiba National Park) located on the northern coast of Rio de Janeiro State, Brazil (October 2004) and was identified by Dr. Marcelo Guerra Santos. The dried specimens were deposited in the herbarium of the Faculdade de Formica de Professors, UERJ (M. Guerra Santos 1.824) and of the Museum National, UFRJ (M. Guerra Santos 1.406). Extraction of the essential oil The essential oil was obtained by steam distillation (1.37 kg of fresh plant) during 4 h in a Clevenger-type apparatus (yield 0,42% v/w), and stored at a low temperature until tested and analysed. Gas chromatography spectrometry analysis.
Essential oil was analysed by a SHIMADZU gas chromatograph equipped with a mass spectrometer using electron ionization. The gas chromatographic (GC) conditions were as follows: injector temperature, 260°C; FID temperature, 280°C; Helium gas , flow rate 1 mL/min and split injection with split ratio 1:40. The temperature from the oven was initially 60°C and then rose to 240°C at a rate of 3°C/min. One microliter of each sample, dissolved in CH2Cl2 (1:100 mg/?L), was injected at ZB5MS column (i.d. = 0.25 mm, length 30 m, film thickness = 0.25 mm) column was used. The mass spectrometry (MS) conditions were ionization Oliveira et al. Chemical and biological investigations of Pilocarpus spicatus essential oils.
The percentage composition of the oils was computed by the normalization method from the GC peak areas. The identification of compounds was performed by comparison of Kovat’s index (KI), determined relatively to the retention times of a series of nalkanes, with corresponding reference data  and MS fragmentation pattern was checked with NIST mass spectra libraries.
Microbial strain Staphylococcus aureus and Escherichia coli which comes from the culture collections of the Lab oratorio de Control Microbiologic, were used for the antibacterial activity experiments. Overnight cultures were prepared by inoculating approximately 2 mL Tryptic soy broth  with 2-3 colonies of each organism. Bacterial strains were cultured overnight at 37ºC. Inocula were prepared by diluting overnight cultures in saline to approximately 108 CFU/mL.
Antibacterial activity Antimicrobial tests were carried out by disk diffusion method  Briefly, a suspension of microorganism was spread on the solid media plates of Tryptic soy agar (TSA; Oxoid). The disks (6 mm in diameter) were impregnated with the essential oil until saturation was reached and placed on the inoculated agar. Vancomycin (30 mg) and ampicillin (30 mg) were used as positive reference standards of the test. The inoculated plates were incubated at 37ºC for 24 h. Antimicrobial activity was evaluated by measuring the zone of inhibition against the test organisms. Each experiment was repeated three times.
TLC assay for acetyl cholinesterase inhibitors TLC assay for acetyl cholinesterase inhibitors was done as described by Marston et al. (2002) with modifications. Sample of essential oil was dissolved in toluene (1:1) and 1?L was applied on the silica gel Alugram SIL G UV254 for TLC (MACHEREY – NAGEL). Physostigmine (Sigma, 1?L at 5mM) was used as positive control. After the chromatographic run of the plate with toluene-ethyl acetate mixture (93:7), it was sprayed with DTNB/ATCI (Sigma) reagent (1:1 solution of 0.4 mm 5, 5’-dithiobis (2- nitro benzoic acid) in phosphate buffer (pH 7.4) and 2.0 mm acetylthiocholine iodide in water) until saturation of the silica. The plate was allowed to dry for 5 min, and then 1.5 U/mL of enzyme suspension of the rat brain was sprayed. After a while, a yellow background appeared, with white spots showing the inhibitory compounds. To confirm the result, it was done another plate under the same conditions . It was used 1?L of p-anisaldehyde ten pecent as a false inhibitor compound.
Afterwards the final solution was applied and after thiocholine solution obtained from enzymatic hydrolysis. Acetyl cholinesterase origin Acetyl cholinesterase enzyme suspension of the rat brain was obtained as it described to Cunha Bastes et al (1991) and Lima et al (1996) with modifications of Maura (1998). Brine shrimp lethality test the protocol established by McLaughlin and Rogers (1998) was employed with modifications. Essential oil dilutions at 50, 10, 1 and 0.1 mg/mL were prepared in DMSO and 50?L were transferred to vials. Seawater (5 mL) was added to each vial, resulting in final concentrations of 1000, 500, 100, 10 and 1 mg/mL, respectively. 2nd instar larvae of ten per vial were added. After 24 hours, all the people that survived were counted and the LC50 added up using the Spearman method.
Positive control test was done using sodium lauryl sulphate. All these experiments were performed in triplicate. Statistical analysis all experiments were performed in triplicate. The mean, standard deviation and coefficient of variation (CV) of the three experiments were determined. The CV values of 15.0 or height were considered statistically significant. The VC values were calculated using the Microsoft Excel program. RESULTS The chemical composition of P. spicatus essential oil was analysed by GC/MS and 17 components were identified, as shown in Table 1. The major components were limonene (41.87%), 2- undecanone (11.0%) and sabinene (10.78%). Oliveira et al. Chemical and biological investigations of Pilocarpus spicatus essential oils Latin-American y del Caribe de Plants The in vitro antibacterial activity of P. spicatus essential oil in comparison with the reference standard included in the study, are shown in Table 2. All the tested strains were sensitive to the essential oil. The differences in inhibition zones diameters of S. aureus (CV = 5.0%) and E. coli (CV = 3.9%) were not statistically significant. Table 2.

Antibacterial activity of Pilocarpus spicatus essential oil Microorganisms Essential oil Antibiotics DDa Vancomycinb Ampicillin Staphylococcus aureus ATCC25923 12 20 nt Escherichia coli ATCC36298 14 nt 32 a Zone of inhibition (mm). NT – not tested. b Vancomicin 30 (mg/disc), ampicillin 10 (mg/disc). In the chromatographic assay for acetyl cholinesterase inhibitors, two white spots were seen, one on the base (false positive) and other on the middle of the plate (a true inhibitory result). P. spicatus essential oil showed toxicity against brine shrimp nauplii (Artemia saline L.) with a lethal concentration 50% (the concentration of test compound that kills 50% of A. saline) value of 3.98 mg/mL. Positive control test was done using sodium lauryl sulphate whose LC50 is approximately 22 mg/mL and the blank test (50 ?L DMSO) no lethality of brine shrimp was observed. DISCUSSION P. spicatus essential oil from Rio de Janeiro State presents monoterpenoids, sesquiterpenoids and aliphatic ketones. Regarding the previously reported chemical composition of many essential oils of Pilocarpus genus has been described (Craveiro et al., 1979; Santos et al., 1997; Andrade-Net et al., 2000, 2002).

However, here we note that (Z)-?-ocimene and viridiflorene were detected for first time in P. spicatus. About this, it is necessary to point out that environmental factors strongly influence the chemical composition of essential oil (Kaastra, 1982) P. spicatus essential oil collected in the state of Ceará (Brazil) displays in vitro antibacterial activity against Pseudomonas aeruginosa and Staphylococcus aureus but no activity against E. coli was reported (Santos et al., 1997). It is not clear whether the antibacterial effect may be caused mainly by a single active constituent or by the combined action of the many active constituents found in essential oil. Terpenoids are active against bacteria but the mechanism of action of terrenes is not fully understood although they suggests  membrane disruption by lipophilic compounds.
It has been reported that essential oil with high terpenoids percentages was probably more effective, as a consequence of higher specificity of the assay for lipophilic compounds. Quite a lot of monoterpenes are known to contain Ache (Houghton et al, 2006). The principal monoterpene identified in P. spicatus essential oil is limonene (41.87%), which is known to be an inhibitor of Ache (Miyazawa et al, 1997). In this oil, there are others monoterpenes that have anticholinesterase activity as ?-Pentene, ?-Pentene (Miyazawa and Yama Fuji, 2005), ?-Trepanned Oliveira et al. Chemical and biological investigations of Pilocarpus spicatus essential oils Latin-American y del Caribe de Plants Medicines y Aromatics Vol.9 (3) 2010 | 210 (Miyazawa et al, 1997), ?-Trepanned (Perry et al, 2002) and sesquiterpene alcohol elemi (Miyazawa et al, 1997). P. spicatus essential oil showed LC50 = 3.98 mg/mL that is a value less than 1000 ?L/mL, which suggests good cytotoxic activity potential and indicating that they may possess a significant antitumor activity since McLaughlin et al (1998). The cytotoxic activity showed the P. spicatus essential oil should be more studied as regards a possible antitumor activity.
CONCLUSION The present study is the first report of (Z)-?- ocimene and viridiflorene for P. spicatus essential oil. In addition, showed antibacterial activity against Gram-negative (E. coli) and Gram-positive (S. aureus), anticholinesterase activity and cytotoxic activity in the BST. So, P. spicatus essential oil might represent a valuable source for pharmaceutical applications.