Leaf Essential Oils of Four Piper Species from the State of Ceará-Northeast of Brazil

Os óleos essenciais obtidos por hidrodestilação das folhas de quatro espécies de Piper (P. arboreum, P. crassinervium, P. dilatatum and P. tuberculatum), coletadas no estado do Ceará, foram analisados por CG-EM. Os rendimentos dos óleos, calculados sobre o peso do material fresco, variaram entre 0,03 – 0,11% (p/p). Os óleos foram caracterizados pela presença de monoe sesquiterpenos, exceto o óleo de P. arboreum, no qual foram identificados apenas sesquiterpenos. β-Elemeno (0,58-3,03%), (E)-cariofileno (2,71-37,78%), germacreno D (3,43-11,81%), biciclogermacreno (2,83-25,07%) e δ-cadineno (0,52-2,44%) foram detectados em todas as amostras analisadas. Os monoterpenos majoritários identificados foram α-pineno (11,27%), β-pineno (20,01%), 1,8-cineol (10,81%) e linalol (28,61%) para P. crassinervium, e α-felandreno (22,53%) e ∆-3careno (10,20%) para P. dilatatum. Biciclogermacreno (25,03 e 25,07%) para P. arboreum e P. dilatatum, (E)-nerolidol (11,12%) para P. arboreum, germacreno D (11,81%) para P. tuberculatum e (E)-cariofileno (10,26 e 37,78%) para P. dilatatum e P. tuberculatum, foram os principais sesquiterpenos. Este trabalho descreve, pela primeira vez, a composição química dos óleos essenciais de P. crassinervium e P. tuberculatum.


Introduction
The genus Piper (Piperaceae) has been recently revised. 1 With approximately 700 species, represented by herbs, shrubs and trees it is widely distributed in the tropical and subtropical regions of the terrestrial globe.][11][12][13][14][15][16] Pursuing the determination of the chemical composition of essential oils from aromatic and medicinal plants from Northeast of Brazil flora, especially from the State of Ceará, we are reporting now the results of the investigation of four Piper species: P. dilatatum L.C. Rich, P. arboreum Aubl., P. tuberculatum Jacq.and P. crassinervium Kunth.Although the non-volatile constitution of all four species have already been reported, 2,6,[17][18][19] the essential oils of P. tuberculatum and P. crassinervium are being reported for the first time.During referee assessment of this paper a report of the essential oils from eleven different plant species, including P. crassinervium, came out in the literature. 20

Results and Discussion
The results for the leaf essential oil analysis of four Piper species are described in Table 1.A total of 52 volatile constituents were identified, accounting for 88.49 -98.62% of the chemical composition of the correspondent oils.All oils showed some similarity in the qualitative composition, but they differ significantly from a quantitative point of view.The essential oils of P. arboreum and P. tuberculatum are characterized by the presence of sesquiterpenes, 88.49 and 81.07%, respectively; while a high percentage of monoterpenes was detected in the oil of P. crassinervium (74.55%).On the other hand, a similar contribution of monoterpenes (42.14%) and sesquiterpenes (54.19%) was observed to the oil of P. dilatatum.As can be seen from Table 1, monoterpenes were not detected in the essential oil of P. arboreum, to which the most significant constituents were the sesquiterpenes bicyclogermacrene (25.07%), (E)-nerolidol (11.12%) and (E)-caryophyllene (8.35%).Previous studies on leaf essential oils of what seems to be three varieties of P. arboreum have been reported and significant differences were found in their chemical compositions, although all them were dominated by sesquiterpenes. 10,11,15The leaf essential oil of P. arboreum Aublet var.latifolium (C.DC) Yuncker from Rondônia, was characterized by an higher content of germacrene D (72.87%), 10 while in the leaf oil of P. arboreum var.arboreum Yunck.from Rio de Janeiro, the most abundant constituents were γ-eudesmol (14.61%) and α-eudesmol (12.21%), followed by bulnesol (8.13%) and (E)-caryophyllene (6.40%). 15On the other hand, in the study of P. arboreum var.latifolium oil from Panama, δ-cadinene (25.80%) and α-copaene (7.40%) were the main compounds. 11omparison of those major constituents for all P. arboreum oils from different origins revealed that all components are present in the oil from Rio de Janeiro, just two of them (germacrene D and α-copaene) are present in the oil from Rondônia and, that the same compounds (germacrene D, (E)-caryophyllene, δ-cadinene and α-copaene) are qualitatively but not quantitatively, present in both oils from Ceará and Panama.In the essential oil of P. dilatatum, from Ceará, α-phellandrene (22.53%) and ∆-3-carene (10.20%) were the major monoterpene hydrocarbons, while bicyclogermacrene (25.03%) and (E)-caryophyllene (10.26%) were the main sesquiterpene hydrocarbons.Earlier investigation of the leaf essential oil of P. dilatatum from Rio de Janeiro, showed that the monoterpenes myrcene (41.70%) and α-pinene (17.70%) were the major components. 15The major mono-and sesquiterpenes of the oil from Ceará are absent, or in very low percentage, in the oil from Rio de Janeiro.The same thing happens with the major monoterpenes of the oil from Rio de Janeiro.The oil of P. crassinervium, rich in monoterpenes, was characterized by a high content of linalool (28.61%), β-pinene (20.01%), α-pinene (11.27%) and 1,8-cineole (10.81%).Recently, the essential oil of P. crassivervium from Ecuador has been examined and was found to be rich in monoterpenes, including α-pinene (15.17%) and β-pinene (10.0%), in accordance with our analysis, but also rich in limonene (26.6%) and α-terpinene (7.79%), 20 not present in the oil from Ceará.All these facts would be maybe attributed to the edafoclimatic conditions related to the different environments in which the plants grown.The most significant constituents of the oil of P. tuberculatum were the sesquiterpenes (E)-caryophyllene (37.78%) and germacrene D (11.81%), while α-pinene (4.06%) and β-pinene (4.51%) were the main constituents of the monoterpene fraction.Unfortunately no other source of P. tuberculatum oil was found in the literature for comparison.

Isolation of the essential oils
Fresh leaf samples of all species were subjected to hydrodistillation for 2 hours in a Clevenger-type apparatus.The isolated essential oils were dried over anhydrous sodium sulfate and, after filtration, maintained under refrigeration before analysis.The yields (m/m) of the oils were calculated based on the fresh weight of the plant materials: P. dilatatum (600 g, 0.07%), P. crassinervium (290 g, 0.11%), P. arboreum (700 g, 0.07%) and P. tuberculatum (660 g, 0.03%).

GC-MS conditions
GC-MS analysis was carried out on a Hewlett-Packard Model 5971 GC/MS using a DB-5 fused silica capillary column (30 m x 0.25 mm i.d.x 0.25 μm film thickness); helium as the carrier gas, flow rate of 1 mL min -1 and with split ratio 1:30.The injector temperature and detector temperature was 250 ºC and 200 ºC, respectively.The column temperature was programmed from 35 °C to 180 ºC at 4 ºC min -1 and then from 180 °C to 250 ºC at 10 ºC min -1 .Mass spectra were recorded from 30 -450 m/z.

Compound identification
The volatile components were identified by comparison of their 70 eV mass spectra with those of the spectrometer data base using the Wiley L-built library and other two computer libraries MS searches using retention indices as a preselection routine. 21,22The identifications were confirmed by comparison of the fragmentation pattern and their retention indices with those reported in the literature. 23,24

Table 1 .
were harvested at the flowering stage from Guaramiranga Mountain (Ceará State, Brazil), in June 2004.P. tuberculatum (# 12985) was harvested from Horto de Plantas Medicinais Prof. Francisco José de Abreu Matos -Universidade Federal do Ceará, in July 2004.All species were harvested from 9 to Chemical composition of four Piper species a a Pa = P. arboreum; Pc = P. crassinervium; Pd = P. dilatatum; Pt = P. tuberculatum; b Compounds are listed in order of their elution from a DB-5 column; c RI = retention indices relative to C 8 -C 26 n-alkanes; d RI = retention indices from literature.