|Pimenta racemosa; 6-tert-butyldimethylsilyl-2,3-diethyl-
β-cyclodextrin; Essential oil composition; Phenylpropanoid; Eugenol;
Chavicol; (S)-(-)-limonene; (R)-(-)-linalool
|The genus, Pimenta (family Myrtaceae), is comprised of about 2 to 5
species of aromatic trees . 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 . 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  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 . 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 .
|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.
|Materials and Methods
|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.
|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 7th edition) and by comparing with the
mass spectral literature data . The relative amounts of individual
components were calculated based on GC peak areas without using
|For chiral GC analysis, a TBDE-β-CD (RESTEK RtTM-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
|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 . 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,
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
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 . The
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
13C values was good. However, the deviations from observed
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.
|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].
|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).
- Bailey LH, Bailey EZ (1976) Hortus. (3rdedn), Macmillan General Reference, New York.
- Bailey LH (1968) Manual of cultivated Plants. The MacMillan Company, New York.
- Neal MC (1965) In Gardens of Hawai'i. Bernice P. Bishop Museum Special Publication 40, Bishop Museum Press, Honolulu, HI.
- Welsh SL (1998) Flora societensis: A summary revision of the flowering plants of the Society Islands. E.P.S. Inc., Orem, Utah 420.
- Institute of Pacific Islands Forestry (2012) Pacific Island Ecosystems at Risk (PIER): Plant threats to Pacific ecosystems: Risk assessments for invasive and potentially invasive species.
- Alitonou, GA, Noudogbessi J-P, Sessou P, Tonouhewa A, Avlessi F, et al. (2012) Chemical composition and biological activities of essential oils of Pimenta racemosa (Mill.) J. W. Moore. from Benin. International Journal of Biosciences 2: 1-12.
- Noudogbessi J-P, Kossou D, Sohounhloue DCK (2008) Chemical and physicochemical properties of essential oils of Pimenta racemosa (Miller) and Chromolaena odorata (L. Robinson) acclimated to Benin. Journal de la Societe Ouest-Africaine de Chimie 13: 11-19.
- Jirovetz L, Buchbauer G, Stoilova I, Krastanov A, Stoyanova A, et al. (2007) Spice plants: chemical composition and antioxidant properties of Pimenta Lindl. essential oils. Part 2: Pimenta racemosa (Mill.) J.W. Moore leaf oil from Jamaica. Ernaehrung (Vienna, Austria) 31: 293-300.
- Ayedoun AM, Adeoti BS, Setondji J, Menut C, Lamaty G, et al. (1996) Aromatic plants from tropical West Africa. IV. Chemical composition of leaf oil of Pimenta racemosa (Miller) J. W. Moore var. racemosa from Benin. J Ess Oil Res 8: 207-209.
- Tucker AO, Maciarello MJ, Adams RP, Landrum LR, Zanoni TA (1996) Volatile leaf oils of Caribbean Myrtaceae. I. Three varieties of Pimenta racemosa (Miller) J. Moore of the Dominican Republic and the commercial bay oil. J Ess Oil Res 3: 323-329.
- Padmakumari KP, Sasidharan I, Sreekumar MM (2011) Composition and antioxidant activity of essential oil of pimento (Pimenta dioica (L) Merr.) from Jamaica. Natural Product Research 25: 152-160.
- Jirovetz L, Buchbauer G, Stoilova I, Krastanov A, Stoyanova A, et al. (2007) Spice plants: chemical composition and antioxidant properties of Pimenta Lindl. essential oils. Part 1: Pimenta dioica (L.) Merr. leaf oil from Jamaica. Ernaehrung (Vienna, Austria) 31: 55-62.
- Adams RP (1995) Identification of Essential Oil Components by Gas Chromatography/Mass Spectroscopy, Allured Publ. Corp., Carol Stream, IL.
- Pragadheesh VS, Yadav A, Singh M, Chanotiya CS (2012) Characterization of volatile components of Zingiber roseum essential oil using capillary GC on modified cyclodextrins. Natural Product Communications, in press.
- Miyazawa M, Hisama M (2001) Suppression of Chemical Mutagen-Induced SOS Response by Alkylphenols from Clove (Syzygium aromaticum) in the Salmonella typhimurium TA1535/pSK1002 umu Test. Journal of Agriculture and Food Chemistry 49: 4019-4025.
- Kubeczka K-H (2002) Essential Oil Analysis by Capillary Gas Chromatography and Carbon-13 NMR Spectroscopy. (2ndedn), John Wiley & Sons Ltd., England.
- Vassão DG, Gang DR, Koeduka T, Jackson B, Pichersky E et al. (2006) Chavicol formation in sweet basil (Ocimum basilicum): cleavage of an esterified C9 hydroxyl group with NAD(P)H-dependent reduction. Org Biomol Chem 4: 2733-2744.
- Bisht D, Chanotiya CS, Rana M, Semwal M (2009) Variability in essential oil and bioactive chiral monoterpenoid compositions of Indian oregano (Origanum vulgare L.) populations from northwestern Himalaya and their chemotaxonomy. Industrial Crops and Products 30: 422-426.
- Konig WA (1998) Enantioselective capillary gas chromatography in the investigation of stereochemical correlations of terpenoids. Chirality 10: 499-504.