Volatiles of Black Pepper Fruits (Piper nigrum L.)

Black pepper (Piper nigrum) is historically one of the most important spices and herbal medicines, and is now cultivated in tropical regions worldwide. The essential oil of black pepper fruits has shown a myriad of biological activities and is a commercially important commodity. In this work, five black pepper essential oils from eastern coastal region of Madagascar and six black pepper essential oils from the Amazon region of Brazil were obtained by hydrodistillation and analyzed by gas chromatography-mass spectrometry. The major components of the essential oils were α-pinene, sabinene, β-pinene, δ-3-carene, limonene, and β-caryophyllene. A comparison of the Madagascar and Brazilian essential oils with black pepper essential oils from various geographical regions reported in the literature was carried out. A hierarchical cluster analysis using the data obtained in this study and those reported in the literature revealed four clearly defined clusters based on the relative concentrations of the major components.


Introduction
Genus Piper (Piperaceae) is represented by about 1500-2000 species of perennial evergreen climbing, lianescent herbs or shrubs distributed in tropical and subtropical regions. Pepper (Piper nigrum L.) is one of the oldest and most extensively used spices and traditional medicines known to mankind. The plant is believed to have originated in India and Indonesia, and has been cultivated throughout the tropical regions [1][2][3]. India, Brazil, Indonesia, Malaysia, Vietnam, and Sri Lanka are the major countries of P. nigrum production [3,4]. The plant can reach up to 50-60 cm in height [5] and is characterized by its simple, alternate leaves, with a few rare cases of opposite or verticillate leaves [1]. The most commonly used part of the plant is the aromatic fruit. Interestingly, white, green, and black peppers are products of the P. nigrum fruits at different ripening stages [3]. White pepper is obtained from the fully ripened fruits after removing the outer skin, green pepper is the unripe fruits, and black pepper is collected before full maturity of the fruit [1,3]. Black pepper has a stronger flavor compared to white pepper while green pepper is characterized by its fresh and herbal flavor. The alkaloid piperine is responsible for the pungent flavor of black pepper [3].

Essential Oils
Volatile oils from commercial suppliers were obtained from the collections of the Aromatic Plant Research Center (APRC, Lehi, UT, USA). The samples from Pará state were provided by EMBRAPA Amazônia Oriental (Brazilian Agricultural Research Corporation) and obtained by hydrodistillation in a Clevenger apparatus (100 g, 3 h). The oils were dried over anhydrous sodium sulfate and their yields calculated from the dry weight of the plant material.

Gas Chromatographic-Mass Spectral Analysis
The essential oils obtained from APRC were analyzed by gas chromatography-mass spectrometry (GC-MS) using a Shimadzu GCMS-QP2010 Ultra operated in the electron impact (EI) mode (electron energy = 70 eV), scan range = 40-400 atomic mass units, scan rate = 3.0 scans/s, and GC-MS solution software. The GC column was a ZB-5 fused silica capillary column with a (5% phenyl)-polymethylsiloxane stationary phase and a film thickness of 0.25 µm, a length of 30 m, and an internal diameter of 0.25 mm. The carrier gas was helium with a column head pressure of 552 kPa and flow rate of 1.37 mL/min. The injector temperature was 250 • C and the ion source temperature was 200 • C. The GC oven temperature was programmed for 50 • C initial temperature, then temperature was increased at a rate of 2 • C/min to 260 • C. A 7% w/v solution of the sample was prepared in dichloromethane and 0.1 µL was injected with a splitting mode (30:1).
Qualitative analysis of the Belém samples was carried out by gas chromatography-mass spectrometry (GC-MS) (Shimadzu QP2010 plus instrument, Shimadzu Scientific Instruments, Columbia, MD, USA) under the following conditions: Rtx-5MS silica capillary column (30 m × 0.25 mm film thickness, (Phenomenex, Torrance, CA, USA); programmed temperature, 60-240 • C (3 • C/min); injector temperature, 200 • C; carrier gas, helium, adjusted to a linear velocity of 1.2 mL/min; injection type, splitless; split flow was adjusted to yield a 20:1 ratio; septum sweep was a constant 10 mL/min; EIMS, electron energy, 70 eV; and temperature of the ion source and connection parts, 200 • C. The retention indices were calculated for all the volatile constituents using a homologous series of n-alkanes (C 8 -C 32 , Sigma-Aldrich). Identification of the oil components was based on their retention indices and by comparison of their mass spectral fragmentation patterns with those reported in the literature [23], and our own in-house library [24]. The component percentages are based on peak integrations without standardization.

Conclusions
The essential oils of black pepper have been analyzed by GC-MS. The oils were dominated by monoterpene hydrocarbons. Black pepper oils from various geographical locations have shown qualitative similarities with differences in the concentrations of their major components. β-Caryophyllene, limonene, β-pinene, α-pinene, δ-3-carene, sabinene, and myrcene were the main components of P. nigrum oil. The cluster analysis revealed four clearly defined clusters for P. nigrum.