Microclimatic Factors and Phenology Influences in the Chemical Composition of the Essential Oils from Pittosporum undulatum Vent . Leaves

O óleo essencial das folhas de um espécime brasileiro de Pittosporum undulatum Vent. foi analisado através de cromatografia a gás-espectrometria de massas (CG-EM), além de análise por RMN após separação cromatográfica. As folhas de P. undulatum foram coletadas durante um ano (Janeiro, Março, Maio, Julho, Setembro e Novembro de 2004) e os óleos essenciais obtidos foram submetidos à análise por CG e CG/EM. O óleo mostrou-se composto por hidrocarbonetos monoterpênicos e sesquiterpênicos, sendo o (+)-limoneno o constituinte principal. Através dos resultados obtidos por essa análise, observou-se uma variação significativa na proporção relativa de (+)-limoneno em cada uma das coletas realizadas, a qual poderia estar correlacionada a parâmetros microclimáticos (temperatura e índice pluviométrico) além de fenologia da espécie estudada.


Introduction
Pittosporum undulatum (Pittosporaceae), named "pauincenso" in Brazil because its leaves and fruits present a characteristic smell, is a tree of 12 m in its natural habitat but usually smaller in cultivation (7-10 m).Small, white, fragrant flowers occur in terminal clusters in spring and early summer and are followed by orange-tan berries 1 cm in diameter in autumn, which persist for several months.This species has been found as a wild plant in tropical forest from Africa, Asia and New Zealand and have been also planted as ornamental specie in other tropical regions of the world such as Brazilian cities, for example São Paulo. 1,2[5] The essential oils from several species of Pittosporum have previously been described.The oil from leaves of P. senacia collected in Madagascar was composed by 20.4% of monoterpenes, 31.7% of sesquiterpene hydrocarbons and 37.8% of sesquiterpene alcohols being the major compounds δ-cadinene (11.3%), α-muurolol (15.9%) and α-cadinol (19.0%). 6From leaves of P. balfourii, collected in Island of Rodrigues, east of Madagascar, was obtained an essential oil composed by 86.2% of monoterpenes and 4.9% of sesquiterpenes, and the major component was identified as myrcene (47.5%). 7In the essential oil from leaves of P. viridiflorum, the occurrence of monoterpenes, hydrocarbon sesquiterpenes and oxygenated sesquiterpenes were reported being δ-cadinene (10.6%) and α-cadinol (18.3%) the main constituents. 8Recently, an extensive study with essential oils from several species of Pittosporum collected on New Zealand have been described. 9The oils from P. crassifolium, P. fairchildii, P. tenuifolium and P. umhellatum was constituted mainly by sesquiterpenes and monoterpenes in low abundance.Other species such as P. anomalum, P. eugenioides and P. dallii were also studied and their essential oils are rich in sesquiterpene alcohols besides esters such as octyl and phytyl acetate. 9he essential oil from leaves of P. undulatum has been subject of one published research in which the plant material was collected in San Miguel Island (Azores).This report describes that 17 compounds were identified and the relative amount of each class of compounds have been described.Monoterpenes (1.6%), hydrocarbon sesquiterpenes (61.5%), sesquiterpene alcohols (16.5%), diterpene (10.7%) and alkanes (6.5%) were found, being the main constituents the sesquiterpenes calamenene (41.4%), farnesol (10.9%), spathulenol (5.6%) and β-selinene (5.2%) and the diterpene (8β,13β)-kaur-16-ene (10.7%). 10n continuation with our studies on essential oils from aromatic Brazilian species, the present investigation reports the chemical composition of the essentials from leaves of P. undulatum from Brazil, and compares the difference in the oil content and composition of other Pittosporum species.Despite of the chemical composition of essential oil from P. undulatum had been described previously, the effect of the harvesting period in the oil composition had not been reported in the literature.
Effects of environmental factors on the variation on the chemical composition of essential oils were observed in several species such as Virola surinamensis, 11 Santolina rosmarinifolia 12 and Helichrysum italicum. 13Therefore, we describe here the analysis of the relative percentage of the leaves oils components of this species obtained in six different periods of one year (January, March, May, July, September and November) and a possible relationship with the microclimatic conditions and phenological state of the studied species.

Plant material
The leaves of Pittosporum undulatum Vent.(Pittosporaceae) were collected in the Universidade Presbiteriana Mackenzie Campus, São Paulo, SP, on January, March, May, July, September and November, 15 th , 2004, in four times during a day (8 a.m., 12 a.m., 4 p.m., 8 p.m.).The leaves were harvested before (January, March, May and July), during (September) and after (November) the flowering stage.The specie was identified by Prof. Dr. Lúcia Rossi from Instituto de Botânica/SP and a voucher specimen was deposited at the Herbarium of the Prefeitura Municipal de São Paulo (PMSP) under code number 8767.

Essential oil distillation
The fresh leaves were hydro-distillated for 4 h in a Clevenger type apparatus.The essential oils were separated from water using CH 2 Cl 2 as solvent, dried over anhydrous Na 2 SO 4 and stored at 4 °C in the dark.For chromatographic separation of the oil components, the leaves were collected on July 15 th , 2004 (500 g) and submitted to the same extraction procedure described above, to give 120 mg of crude essential oil (yield 0.02%).

Microclimatic factors
The registered values of temperature and relative humidity have been measured in situ with a digital Pocket Weather Meter Kestrel 3000 (Nielsen-Kellerman -USA).Precipitation values have been registered using a pluviometer made in our laboratory (values in millimeters) from 12 th to 18 th day in each collection.

Gas chromatography analysis (GC)
The crude essential oils were analyzed by GC with a Hewlett-Packard 5980 Series II system using an HP-5 column (30m × 0.32 mm internal diameter, with 0.25 μm film thickness) and helium as carrier gas (1 mL min -1 ).The oven temperature was kept at 60 °C and programmed to 280 °C at a rate of 3°C min -1 and kept constant at 280 °C for 10 min.The injector temperature was at 220 °C and detector (FID) at 280 °C.The percentage compositions were obtained from electronic integration measurements using flame ionization detector (FID).A serie of linear nalkanes was used as reference points in the calculation of relative Kovats indices (KI).

Gas Chromatography -Mass Spectrometry analysis (GC-MS)
GC-MS analysis was carried out in a Shimadzu GC-17A chromatograph interfaced with a MS-QP-5050A mass spectrometer.Helium was used as the carried gas.The MS operating conditions were: ionization voltage 70 eV, ion source 230 °C.The GC analysis was done with a DB-5 column (30m × 0.25 mm internal diameter, with 0.25 μm film thickness) and the operating conditions were identical with those of the GC analysis.
Retention indices for all compounds were determined according to the Kovats method relative to the linear nalkanes series (C 8 to C 20 ).The identification of the compounds was done by comparison of Kovats indices and by matching their fragmentation patterns in mass spectra with those of standard compounds and published mass spectra data 14 along with mass spectra of authentic compounds.

Chiral phase-Gas Chromatography analysis (GC)
The enantiomeric excess of limonene was determined by GC in a Shimadzu GC17A system using a Varian W cot fused silica coated with CP Chirasil-dex CB (25 m × 0.25 mm internal diameter, with 0.25 μm film thickness) as stationary phase and helium as carried gas (1 mL min -1 ).The oven temperature was kept at 60 °C and programmed to 280 °C at a rate of 5 °C min -1 and kept constant at 280 °C for 5 min.The injector temperature was at 220 °C and detector (FID) at 280 °C.

Results and Discussion
The crude essential oils from leaves of Pittosporum undulatum were analyzed by GC (DB-5 capillary column) and GC-MS associated to determination of the Kovats indexes.The oil contained ten identified components, corresponding to monoterpenes (β-pinene, β-myrcene, limonene, δ-elemene) and sesquiterpenes (α-copaene, β-elemene, β-caryophyllene, aromadendrene, bicyclogermacrene, δ-cadinene), being limonene the main constituent.Literature data indicated that (+)-limonene showed insecticidal activity against Rhyzopertha dominica (F.), lesser grain borer, and Tribolium castaneum (Herbst), red flour beetle, which are important pests of stored grain. 19dditionally, the crude oil was fractionated by SiO 2 / AgNO 3 column followed by GC analysis, which indicated that fractions 3-7 were composed by a pure component (99.7%).These fractions were pooled together and submitted to chiral gas chromatography analysis, measurement of specific optical rotation, LREIMS and 1 H and 13 C NMR spectrometry to confirm the identity of the main component as (+)-limonene, in comparison with literature data. 18,19o verify the accumulation of this monoterpene as major component, the crude oils of P. undulatum were obtained from leaves collected on the 15 th day in January, March, May, July, September and November, 2004 (four samples in each collection) and submitted to GC and GC/ MS analysis.The medium values of components in the essential oils obtained from each collection, in the six months of analysis, corresponding to monoterpenes (68±10 -83.9±0.6%) and sesquiterpenes (7±1 -11±1%), as showed in Table 1.The yields of the essential oils appear in Table 2, along with registered precipitation, relative humidity, temperature values and phenology of the analyzed specimen.
We have detected quantitative but not qualitative variations on the yields and chemical constituents of the essential oil during the period of study.The yields of the essential oils were constant when the leaves were collected in the specimen sterile period (0.02%), and increase in the flowering and fruiting stages (0.05 -0.06%, respectively).
During the sterile period (January, March, May and July) the relative level of (+)-limonene, was not constant (67±10 to 80.8±0.4%), which could be related to microclimatic factors such as temperature, air relative humidity and precipitation. 19As showed in Table 1, in March and July the relative proportion of (+)-limonene was lower (67±10 and 69±3%, respectively) in comparison with other collection periods.In these months the precipitation values were high (33 and 65 mm, respectively) which suggest an influence of this microclimatic factor in the accumulation of (+)-limonene in the crude essential oil. 12However, in September and November (78±6 and 76±6%, respectively), in the flowering and fructification periods, the relative amount of (+)-limonene was higher than March and July (67±10 and 69±3%, respectively) despite of the high precipitation index in November (39 mm).High values of limonene in the essential oil from leaves of Helichrysum italicum in flowering stage were observed previously. 13hese data suggest that in the flowering and fructification periods the production of more volatile derivatives was intensified, in agreement to literature data. 11,12esides, a relationship between production of more volatile compounds and air relative humidity has been observed. 20In the present study we detected a higher production of monoterpenes, mainly (+)-limonene, in a dry season (January) when the air relative humidity was 64 ± 5% (sterile period).
Therefore, although precipitation, temperature and relative humidity might be expected to affect the oil chemical composition, it also could be partially depend on the phenological state. 22t is important to mention that in the P. undulatum leaves oil previously analyzed by Mananjarasoa et al. 6 was observed a predominance of hydrocarbon sesquiterpenes, being calamenene the main constituent (41.4%), and a low amount of oxygenated derivatives.In the present work the main constituents were the hydrocarbon monoterpenes, being (+)-limonene the major component.The occurrence of one monoterpene (myrcene 47.5%) as the most abundant component has been described previously by Gurib-Fakim and Demarne, 7 in the essential oil from the leaves of P. balfourii.Therefore, the composition of the essential oil of P. undulatum collect in Brazil is quite different from the oils extracted from others Pittosporum species, which should be associated to several environmental factors.