Chemical composition and antinociceptive activity of the essential oil of Piper mollicomum and Piper rivinoides

The analysis of chromatographic profiles of the essential oil of Piper mollicomum and Piper rivinoides for Multidimensional gas chromatography (MDGC) allowed the identification of 18 compounds in essential oil of Piper mollicomum (EOPM) and 19 compounds in essential oil of Piper rivinoides (EOPR), being β-ocimene and α-pinene which are the major compounds, respectively. In the test of writhing induced by acetic acid (0.8%) to verify the antinociceptive activity, the EOPM and EOPR at 1 mg/kg showed significant inhibition of writhing of 20.90 and 50.25%, respectively compared to the control. The aim of this study was to evaluate the chemical composition and antinociceptive activity of essential oils from fresh leaves of P. mollicomum (EOPM) and P. rivinoides (EOPR).


INTRODUCTION
Many essential oils are found to exhibit varied biological properties, such as spasmolytic, anxiolytic, anticonvulsant and antinociceptive activity (Almeida et al., 2003;Santos et al., 2005;Pultrini et al., 2006).These effects are probably due to high structural diversity of the essential oil constituents.The study of each individual chemical component is critical to understand its mechanism of pharmacological action and toxicity, including potentially beneficial clinical effects on human health.Considering that the essential oils and their constituents are common in many plant species and are used in cosmetic and pharmaceutical preparations, as well as in the food industry, it is important to review the pharmacological potential of the essential oil constituents with analgesic-like activity (Dantas et al., 2004;Menezes et al., 2007).
In folk medicine the fruits of P. mollicomum are used to treat stomach problems, and are also widely used in venereal diseases and their roots, when chewed, are used to numb the pain of teeth (Guimarães and Giordano, 2004).P. rivinoides Kunthis known as "ruão" is also native to Brazil.It is found in the Southeast of the country and in the states of Pernambuco and Santa Catarina (Guimarães et al., 2012).Its fruits are eaten by animals (Guimarães and Monteiro, 2006).There are no phytochemical study for P. rivinoides.

Plant
Piper mollicomum Kunth, Piper rivinoides Kunth and Piper aduncum L. were cultivated in Platform of Agroecological Phytomedicines (PAF), at FIOCRUZ/RJ.The supply and tipping species were done by the botanist Sergio Monteiro da Silva, head of Green Pharmacy Herbal "FFAR" and the vouchers were deposited under the codes 550354, 550355 and 550356, respectively.The determination of the species was made by Dr.
Extraction of volatile compounds from Piper species 400 g of the fresh leaves of both species were submitted to steam distillation for 3 h, using a modified Clevenger apparatus to yield 0.5% of P. mollicomum and 0.4% of P. rivinoides in the Laboratory of Natural Products Chemistry, Institute of Technology in Pharmaceuticals (Farmanguinhos), FIOCRUZ, Rio de Janeiro, RJ, Brazil.The essential oil samples were packed in amber vials and stored at -20°C in freezer until analysis.

Analysis and identification of volatile compounds by MDGC
An essential oil/dichloromethane solution 1:20 was prepared to be injected in the Shimadzu MDGC system (Figure 2).This multidimensional gas chromatography (MDGC) system presents two gas chromatographs: GC-2010 (GC1 and GC2) and one quadrupolo MS-QP2010.The MDGC transfer device located in the GC1, is linked to a unit of advanced pressure control (APC) that provides the carrier gas helium (He) at constant pressure.GC 1: an HP-25 m × 0.20 mm FFAP i.d.× 0.33 (Agilent) was used as column; injection mode split (230°C); injection volume: 1.0 µl; He as carrier gas; temperature program: 60°C 1 min, 60 to 80°C to 3°C/min 80 to 250°C and the 20°C/min (1 min); FID 250°C; total flow: 14.8 ml/min.GC 2: It was used as column 230 m × 0.25rtx-5MS mm i.d.× 0.25a (Restek); temperature program: 50°C (6 min), 50 to 150°C to 10°C min and 150 to 250°C (2 min) 5°C/min.MS: Ion source: 250°C; interface temperature: 250°C, scan interval: 40 to 400 m/z; scan speed: 2000 amus.All data were obtained by GC solution software (Shimadzu) to FID (GC1) and by Solution gas chromatography mass spectrometry (GC-MS) software (Shimadzu) MS (GC2).The identification of the constituents was assigned based on comparison of their retention indices and mass spectra with literature data (Adams, 1995;Santos et al., 2001;Mesquita et al., 2005;Lago et al., 2007).Peaks were identified by comparing its mass fragmentation pattern with that of standard compounds as well as with data available spectral library (Wiley NIST Libraries) of the instrument.All analyses were performed in triplicate and quantification of each constituent obtained by standardization of the areas (%).In this study, a retention indice (RI) was adopted using the injection of homologous series of hydrocarbons (C8 to C28), calculated with data obtained in the second dimension for the calculations (GCMS).

Animals
Male Swiss mice weighing 23 to 30 were used.The animals were obtained from the animal-breeding colony at the Oswaldo Cruz Institut Foundation (FIOCRUZ) and maintained in the animal room of the Department of Physiology and Pharmacodynamics, in Oswaldo Cruz Institut (IOC/FIOCRUZ), with free access to pelleted diet and water with period of light/darkness (12/12 h) and temperature (24°C) controlled.The animals were allowed to adapt to the laboratory for at least 2 h before testing and were used only once.The experiments in this study received prior approval from the Oswaldo Cruz Institute Foundation´s Animal Welfare Committee.The license for use of the animals was granted by Center for the Study of Animal Use (CEUA/FIOCRUZ) under number 033/09.After the experimental procedures, animals were euthanized in CO 2 and incinerated.

Antinociceptive effect
The essential oil of Piper rivinoides (EOPR) and essential oil of Piper mollicomum (EOPM) samples were evaluated for their analgesic activity through Collier's test acetic acid induced writhes in mice to evaluate their antinociceptive effect (Collier et al., 1968).Groups of 12 animals were used.The amount of writhings was evaluated for 10 min, beginning the registers after 5 min of the injection of the stimulus (acetic acid 0.8% intraperitoneally).The doses of EOPR and EOPM (1 mg/kg) and the control analgesic, diclofenac (50 mg/kg), were administrated orally (200 µl), always an hour before the induction of pain.The control group was treated with saline solution + 1% dimethyl sulphoxide (DMSO) (diluent).

Statistical analysis
Statistical significance was determined by a one-way analysis of variance, followed by Student´s-Neuman-Keuls test (Prism 4, GraphPad, La Jolla, CA, USA).The results were considered significant for values of p ≤ 0.05.

RESULTS
The EOPM yield was 0.5% and 18 compounds were identified in 94.91% of the obtained oil from fresh leaves.This sample was analyzed through two sequential separations being the first in a polar column and FID detector and the second in a column apolar where the coumpounds were identified.The identified monoterpenes were (E)-ocimene, (Z)-ocimene and linalol, representing 28.59% of total essential oil.The other identified compounds 50.54% are sesquiterpenes not oxygenated and 15.2% are oxygenated sesquiterpenes.
The major compounds from this sample are α-pinene (32.93%), β-pinene (20.74%), caryophyllene (7.6%), germacrene B (6.72%).The EOPM and EOPR composition is presented in Table 1.The antinociceptive activity of P. rivinoides and P. mollicomum leaves essential oil has never been measured (Figure 1).The dose of 1 mg/kg was used following the protocol set out in our research group in the search for bioactive molecules.The antinociceptive activity in mice writhing test is shown in Table 2.This effect was observed in the two sample oils, the EOPR inhibited about 20.90% and the OEPM inhibited about 50.25% of the writhings (Table 2).Oral administration of the EOPR and EOPM at 1 mg/kg dose did not induce any apparent toxic effect.No behavioral alteration, lesions or gastric bleeding was observed.

DISCUSSION
The income found for the essential oils studied is within the range found in other species of Piper.Mesquita et al. (2005) extracted the essential oil from nine Piper species and they found 0.3 to 0.8% of yield.Potzernheim et al. (2006) obtained 0.3, 0.4 and 0.3% of the essential oil  from P. arboretum, P. dilatatum and P. hispidum, respectively.Carmo et al. (2012) in their work about the essential oil from P. demeraranum and P. duckei obtained 0.6 and 0.5% of yield.This is the first chemical study of the essential oil from P. rivinoides Kunth.However, the data of the characterized compounds conformed to those described for the Piper genus (Table 3).Costantin et al. (2001) highlighted as major consti-tuents α and β pinene from the P. cernuum oil.Potzernheim et al. (2006) also found these compounds as major constituents of the essential oil from P. hispidum.The P. crassinervium oil are rich in monoterpenes and it presents high amounts of linalol, βpinene and α-pinene (Cysne et al., 2005).Mesquita et al. (2005) reported that the essential oil of P. vicosanum and P. submarginalum are comprised mostly of monoterpenes and α-pinene and β-pinene.These results corroborate with those presented in this study.
The retention-index concept is a valuable, frequently used tool in GC to support compound identification, especially for essential oils.Calculation of the 1D retention index is relatively straightforward, these values change with the MDGC.In the present study we have adopted a retention index using the obtained data injection of an alkanes series in the second dimension (Marriott et al., 2012).The EOPR and EOPM possibly have antinociceptive activity on the central and peripheral nervous system.This activity was demonstrated in the inhibitory effect of mice writhings (Table 2 and Figure 1).The nociceptive response to acetic acid may involve a direct stimulation of nociceptive afferent fibers, due to a pH decrease or a synthesis of inflammatory mediators, such as metabolites of arachidonic acid through cyclooxygenase, with subsequent biosynthesis of prostaglandins (Franzotti et al., 2002).The essential oil tested reduced the writhing, then they possibly exert peripheric and central action by inhibition of endogenous mediators (Collier et al., 1968).
The EOPM showed a high inhibition of writhing compared as used reference drug (diclofenac) which inhibited the writhing in a dose of 50 mg/kg while the essential oil sample inhibited 58.36% of the writhing at 1 mg/kg inhibited 50.26%.Alves et al. (2010) reported that the sesquiterpene α-bisabolol is a peripheral nervous blocker.Guilhon et al. (2011) showed that the essential oil from Lippia gracilis which the major compound is ocimene inhibited significantly the number of writhing in acetic acid-induced contortions model.These data confirm our results, since ocimene and α-bisabolol are also the major compounds of EOPM.

Conclusion
This multidimensional GC×GC-MS configuration analysis provides easy and reliable, flavor and fragrance samples principally identified in many compounds would not be identified using one dimension.In accordance with the results of the essential oils of P. molicomum and P. rivinoides there are active constituents with antinociceptive activity as demonstrated in the employed method, suggesting their potential for therapeutic purposes.

Figure 2 .
Figure 2. Total ion chromatogram of the essential oil P. rivinoides.

Table 1 .
Chemical composition of two Piper species a a EOPM = P. molicomum essential oil; EOPR = P. rivinoides essential oil; c RI = retention indices relative to C 8 -C 28 n-alkanes.

Table 2 .
Results of analgesic activity of EOPM and EOPR.

Table 3 .
Major compounds reported to species of Piper genus.