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Abstract

The chemical composition of the essential oil of ripe berries (11.8% v/w) and leaves (0.9% v/w) of Piper cubeba L. fils. (Piperaceae) was investigated by GC and GC/MS. Sabinene (9.1%), ?-elemene (9.4%), ?-caryophyllene (3.1%), epi-cubebol (4.3%), and cubebol (5.6%) were the main components of the berry oil. trans-Sabinene hydrate (8.2%), ?-caryophyllene (5.0%), epi-cubebol (4.2%), ?-cadinene (16.6%) and cubebol (4.8%) were the main components of the leaf oil. No large qualitative differences were found in the composition between berry and leaf oil, although the berries contained a considerable amount of constituents in traces (

Key Word Index

Piper cubeba, Piperaceae, essential oil composition, ?-cadinene.

Introduction

The genus Piper belongs to the Piperaceae, a family with more than 700 species throughout the tropical and subtropical regions of the world. Piper cubeba, (in Indonesia known as kamukus), is a plant native to Java and Borneo that produces spicy berries (cubeb berries). It is now also cultivated in several other tropical areas, including East Africa. In Indonesia P. cubeba is valued as a medicinal plant (1,2).

Many species of the genus Piper are used in traditional herbal medicine, and have shown antifungal, insecticidal, anthelminthic and antitumor activities. They are also used for the treatment of cough, bronchitis, intestinal diseases and rheumatism (3). A number of polyhydroxy cyclohexanes have been isolated from Piper cubeba and shown to display tumour inhibitory, antileukemic and antibiotic activities (4).

Essential oil investigations of a number of Piper species have been reported, but only one gives a detailed overview of the essential oil of cubeb berries (3,5-8). The aim of the present study was to investigate the oil composition of P. cubeba berries and leaves from Indonesia.

Experimental

Plant material: The ripe berries and leaves of Piper cubeba were collected from Jatiroto, Temmanggung (Central Java, Indonesia) in April 2002 and authenticated at the Department of Biology, Institut Teknologi Bandung (ITB) (Bandung, Indonesia), based on the Flora of Java (9). A voucher specimen (HBGlOPCOl) has been deposited at the Herbarium Bandungense.

Isolation procedure: The oil samples were separately isolated from 20.0 g of air-dried and freshly ground (1 mm) leaves and fruit material by hydrodistillation for 4 h in 300 mL water, according to the determination of the essential oil content in vegetable drugs, using the apparatus described in the Nedetiandse Farmacopee, 6th edn, 2nd printing ( 10). Xylene (100 \iL) was used as the collection liquid, and the oil was stored at -2O0C until analyzed. The oil was diluted 50 times with cyclohexane prior to GC and GC/MS analysis.

In addition, the oil was separated into two fractions with hydrocarbons and oxygen-containing compounds, respectively, by eluting 250 pL of oil on a Bakerbond SPE column, filled with 1 g of silica gel (#7086-07, J.T. Baker, Deventer.The Netherlands), with subsequently 5 mL n-hexane and 5 mL diethyl ether. After gentle evaporation of the solvents ofboth fractions, 50 uL of each residue were diluted with 950 pL cyclohexane and submitted to GC and GC/MS analysis.

GC: GC analysis was performed on a Hewlett-Packard 5890 Series II gas chromatograph equipped with a 7673 injector and a Hewlett Packard 3365 Series II Chemstation, under the following conditions: column, WCOT fused-silica (J & W) DB-5 (30 m x 0.26 mm; film thickness 0.25 µm); oven temperature program, 60°-290°C at 3°C/min; injector temperature, 250°C; detector (FID) temperature, 300°C; carrier gas, He; inlet pressure, 18 psi; linear gas velocity, 31.8 cm/s; split ratio, 56:1; injected volume, 1.0 µL.

GC/MS: A Shimadzu GC/MS QP5000 system was used equipped with a GC-17A gas chromatograph, an AOC-20i auto injector, and GC/MS solution version 1.10 software. The GC conditions were: column, WCOT fused-silica (J & W)DB-5(30m x 0.26 mm; film thickness 0.25 µm); oven temperature program, 60°-240°C at 3°C/min; injector temperature, 275µC; carrier gas, He; inlet pressure, 75 pKa; linear gas velocity, 81.4 cm/s; column flow, 2.5 mL/min; total flow, 56.7 mL/min; split ratio, 21:1; injected volume, 1.0 µL. MS conditions: ionization energy, 70 eV; ion source temperature, 250°C; interface temperature, 250°C; scan speed, 3 scans/s; mass range, 34-350 u.

The identity of the components was assigned by comparison of their retention indices, relative to C^sub 9^-C^sub 22^ n-alkanes, and mass spectral databases and from the literature (11-13). The percentages of the components were calculated from the GC peak areas, using the normalization method.

Results and Discussion

Hydrodistillation of the berries of Piper cubeba yielded 11.8% (w/w) and the leaves 0.9% (v/w) oil. In total 103 components could be identified in the berries, dealing with 59.6% of the oil. In the leaves, 62 components could be identified, corresponding with 77.9% of the oil. As far as we know, this is the first time the oil composition of P. cubeba leaves has been investigated.

In a number of P. cubeba berry oils, Lawrence identified 71 components with ?-cubenene (7-9%), ?-copaene (10-14%), ?-cubebene (7-11%), ?-cadinene (9-10%) and cubebol (9-10%) as the main components. More recently a commercial sample was also analyzed by Lawrence. Its main component was sabinene (30%), whereas cubebol was only present at 5.7% (18).

Comparing the results of our present study with those from the three older reports, we come to the following conclusions. Cubebol is one of the main components in the berry oil, but the amount seems to depend on the origin of the material. Indonesian samples contained about 10%, the Indian and Sri Lankan samples considerably more. We identified cubebol together with epi-cubebol. The other studies do not discriminate between the two epimers. Less abundant in our samples, compared with the previous studies, were ?-cubebene, ?-copaene and ?-cubebene.

Lawrence found cubenol in the berry oil from India (17). We did not find this compound, but detected 1,10-di-epicubenol (trace) and epi-cubenol (0.3%) in the berry oil from Indonesia.

These differences may be used as a tool for the characterization of P. cubeba oils from different origin, although further systematic studies are needed to prove this and correlate this with qualitatively and quantitatively.