The genus, Pimenta (family Myrtaceae), is comprised of about 2 to 5 species of aromatic trees [1]. Pimenta racemosa (Mill). JW Moore (syn. Myrtus caryophyllata, Lacq. Not L, P. acris Kostel) commonly known as Bay or Bay-rum-tree, up to 25 ft high, leaves leathery, obovate or elliptic, finely reticulate veined; flowers white with 5 lobed calyx, is indigenous to West Indies, Venezuela and Guiana and is used in preparation of bay-rum [2]. The plant is similar to allspice (Pimenta dioica) and can be differentiated by the presence of elliptic leaves with fine venation, slightly larger fruit and 5 lobed calyx whereas allspice is 4 lobed [1,3].Welsh reported [4] the plant as 4-12 m tall tree; 3-10 mm long petioles; (1.5) 4-10 (12.5) cm long leaf blades, 2.5-6 cm wide, obovate to oblanceolate or elliptic, coriaceous, obtuse, acute basally, entire, finely reticulately veined, with 5-7 pairs of rather obscure, main lateral veins, shiny above, dull and pale beneath; pedunculate cymes; white flowers; 5-lobed calyx; ovoid fruit, black at maturity [4]. In a Pacific Island Ecosystems (PIER) report, Pimenta has been identified under high risk category, which indicates that the species poses a high risk of becoming a serious pest or may be threat to ecosystems of the Pacific islands [5].

To date, there has been good number of publications on oil compositions from P. racemosa [6-9] and P. dioca [10-12] are available. However, gas chromatography using substituted cyclodextrin for stereochemical/enantiomeric characterization of the P. racemosa terpenoid compounds has not been undertaken for study till date. The present communication reports composition of cultivated P. racemosa oil using GC-FID, enantio-GC-FID, GC/MS and NMR techniques.

Plant material and isolation of essential oils: P. racemosa was collected from CSIR-CIMAP campus, Lucknow during spring and autumn seasons. The essential oil from leaf was extracted by hydro distillation using a Clevenger-type apparatus for 4 h. All oil samples were stored at 4ºC prior to analysis.

GC analysis

A PerkinElmer Auto System XL GC, fitted with an Equity-5 column (60 m×0.32 mm i.d., film thickness 0.25 μm), was used for GC analysis. The column oven was programmed from 70ºC to 250°C at a rate of 3ºC/min, with initial and final hold times of 2 min, and programmed to 290ºC at 6°C/min, with a final hold time of 5 min, using H₂ as carrier gas at a constant pressure of 10 psi, a split ratio of 1:35, and injector and detector (FID) temperatures of 290 and 280°C, respectively. GCMS utilized a Perkin Elmer Auto System XL GC interfaced with a Turbomass Quadrupole mass spectrometer based on the above oven temperature program. Injector, transfer line and source temperatures were 250°C; ionization energy 70 eV; and mass scan range 40-450 amu. Characterization was achieved on the basis of retention time, elution order, relative retention index using a homologous series of n-alkanes (C₆-C₂₈ hydrocarbons, Polyscience Corp. Niles IL), coinjection with standards in the GC-FID capillary column (Aldrich and Fluka), mass spectral library search (NIST/EPA/NIH version 2.1 and Wiley registry of mass spectral data 7^th edition) and by comparing with the mass spectral literature data [13]. The relative amounts of individual components were calculated based on GC peak areas without using correction factors.

Chiral analysis

For chiral GC analysis, a TBDE-β-CD (RESTEK Rt^TM-β-DEXse fused silica capillary columns (30 m×0.25 mm id, 0.25 μm) was used in a Varian CP-3800 gas chromatograph. The oven temperature was programmed from 70°C (hold 3 min) to 120°C at a rate of 3°C/min and 230°C at a rate of 5°C/min. Hydrogen was used as carrier gas at 1.8 mL/min constant flow. Injector and detector temperatures were 220°C and 230°C, while the elution order was confirmed as per the previous reports [14].

NMR report experiment

For NMR experiment, Bruker Avance-300 (300MHz) was utilized for ¹H- and ¹³C-NMR experiments with tetramethylsilane (TMS) as internal standard. About 40 mg of the essential oil was dissolved in CDCl₃ and spectral data are reported. Chemical shifts are reported in ppm units relative to CDCl₃ set to 7.26 (¹H-NMR) and 77.0 (¹³C-NMR) (multiplicity: s, singlet; d, doublet; t, triplet; q, quartet; m, multiplet; br, broad). The identity of compounds was established by comparison of spectral data [15-17].

The oil yield of Pimenta racemosa leaves was 0.02% (w/w). In total, 18 compounds were identified, accounting for 97.2-97.8% of volatile constituents (Table 1; Figure1). Phenylpropanoid contributes major percentage to the oil with eugenol as principal component (72.9-92.9 %; 2). Other minor constituents identified were β-myrcene (0.3-9.6; 1) limonene (0.2-3.8; 5) and chavicol (1.3-7.7; 3) suggesting a high molecular diversity in the essential oil. The total terpenoid proportion recorded was less than 3.9%. A comparison on various published reports on major constituents from genus Pimenta are listed (Table 2). The P. racemosa leaf oil may be distinguished from P. dioica by the presence of high β-myrcene proportion followed by chavicol. Moreover, chavicol may be regarded as marker constituent of P. racemosa leaf oil, which was not reported from natural P. dioica except in one market sample, possibly due to adulteration [12]. On contrary, methyleugenol (4), which contributes good proportion to P. dioica was completely absent in P. racemosa under present study. The characterization of eugenol and chavicol in essential oil was done using ¹H-, ¹³C-NMR and DEPT experiments. In ¹H-NMR, sharp signals for aromatic protons were observed (δ ppm 6.69-6.88, 3H, m) followed by resonances for exocylic double bond (5.06-5.12, 2H, m) and (5.91-6.04, ¹H, m), respectively. The presence of methoxy group (δ 3.88, 3H, s) in the arene ring system was also significant. The methylene protons in C6-C3 side chain was marked by a sharp doublet at δ 3.33-3.35 (d, J=6.3 Hz). In ¹³C-NMR, a total of ten carbon resonances attributed to one methyl, two methylene, four methine and three quaternary carbons were identified for eugenol. The exocylic double bond (δ 137.78, ¹H, δ 115.46, 2H) and one methoxy (δ 55.79) groups were also present (Table 3; Figure S1-S3). Further, the NMR spectral data of eugenol were comparable to the published reports [15-16]. For chavicol, the characteristic aromatic ring proton signals were observed at δ 6.77 (2H, d, J=8.4 Hz); δ 115.20 and δ 7.05 (2H, d, J=8.1 Hz); δ 129.60 in ¹H and ¹³C, respectively. The methylene protons in C6-C3 side chain was marked by a sharp doublet at δ 3.33-3.35 (d, J=6.3 Hz). In ¹³C-NMR, a total of nine carbon resonances attributed to two methylene, five methine and two quaternary carbons were identified. The carbon directly attached to –OH was observed downfield at δ 154 ppm. The NMR spectral data of chavicol were comparable to the published reports [17]. The ¹H and ¹³C chemical shift values for eugenol and chavicol were also calculated and verified using the expressions δ=7.27 + Σ S and δ=128.5 + Σ S, respectively where S represents substitution at ortho-, meta- or para-position. The agreement between the calculated and observed ¹H and ¹³C values was good. However, the deviations from observed ¹H value were greater in eugenol as compared to chavicol because the former possessed ortho substitution. Hence, we conclude that, in addition to the GC-FID, enantio-GC-FID and GC/MS experiments, NMR could be utilized successfully to characterize P. racemosa leaf essential oil.

Figure 1: Major volatile constituents identified in Pimenta racemosa essential oil.

Table 1: Relative percentage of constituents in Pimenta racemosa (Mill.) J.W. Moore essential oil.

Table 2: Comparison of compositions (%) of major constituents in Pimenta essential oil.

Table 3: NMR (δ ppm, 300 MHz) data of Pimenta racemosa essential oil for eugenol and chavicol.

Chiral analysis

Two marker chiral pairs such as limonene and linalool were studied for their enantiomer discrimination (Table 4). Chiral phase coated with 6-tert-butyldimethylsilyl- 2,3-diethyl-β-cyclodextrin revealed enantiomeric excess for (S)-(-)-limonene (41.1-45.3%; Figure 5b) over (R)-(+)- limonene (Figure 5a). Similarly, (R)-(-)-linalool (86.7-89.9%; (Figure 6a) was recorded in high excess as compared to (S)-(+)-linalool (Figure 6b). Besides above, there has been no chiral differentiation observed for other constituents in the leaf oil. Since, phenylpropanoids lack chirality. Therefore, high (R)-(-)-linalool excess may be one of the authenticating tool for P. racemosa essential oil. In conclusion, the systematic chiral investigations have revealed that the presence of both enantiomers is common for monoterpenes such as limonene and linalool in P. racemosa essential oil and the similar signatures had been observed in many earlier reports [18,19].

Table 4: Enantiomeric composition (%) of chiral terpenoids in leaf essential oils of Pimenta racemosa.

The authors are grateful to the Director, CIMAP for the facility and encouragement. This work was funded by the Department of Science and Technology, New Delhi under Fast Track grant SR/FT/CS-036/2010).