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Comparative Analysis of Supercritical CO2 Extract and Oil of Pimenta Dioica Leaves

Posted on: Thursday, 8 September 2005, 03:01 CDT

Abstract

Leaves of Pimenta dioica were used for supercritical extraction with carbon dioxide to isolate the corresponding volatile concentrate. The vegetable fragrances were isolated by supercritical CO2 extraction coupled to a fractional separation technique. The process was carried out operating at 90 bar and 50C in the extraction vessel, at 90 bar and below -15C in the first separator to selectively precipitate the cuticular waxes and at a pressure between 15 and 20 bar and temperatures in the range (15-21C) in the second separator to recover the volatiles. GC/MS analysis of the extract allowed the identification of the volatile concentrate composition. The main constituent in the extract was eugenol (77.9%).

Key Word Index

Pimenta dioica, Myrtaceae, essential oil composition, supercritical carbon dioxide extract composition, eugenol.

Introduction

The genus Pimenta dioica belongs to the Myrtaceae family, it is widespread in Central America, West Indies and Mexico. It was imported in Europa by Colombo; at the beginning it was mistaken for pepper and so-called pimienta. It is said Jamaica pepper because the growings became in 1655 by the English in the tropical climate of Jamaica. The berries of this tropical tree are consumed as a spice and its essential oil has been reported to have antimicrobial, antifungal and insect-repellent properties (1). In fact, eugenol is an attractant of the Japanese beetle Popillia japonica (Scarabaeidae) but a potent repellent of the mosquito Aedes aegypti (Insecta) (2), and also β-caryophyllene another constituent could have contributed to the acaricidal activity of the oil (3). In the present work, we show the results concerning the obtaining of a volatile concentrate from the leaves of P. dioica by means of supercritical carbon dioxide extraction, in a single extraction stage.

Supercritical fluid extraction, SFE, is a valid alternative for the production of flavors and fragrances from natural materials. Compressed CO2 is able to solubilize hydrocarbon and oxygenated mono- and sesquiterpenes (4), the main constituents of essential oils. The separation of the extractant is easy and the volatiles are devoid of residues that pose a risk for human use. Conventional processes such as distillation, solvent extraction etc., often require additional steps such as separating the extractant, and are usually inferior to CO2 with respect to their selectivity. In addition, the lower temperature in the SFE avoids thermal degradation and the low water content limits hydrolysis. Consequently, a volatile concentrate obtained by SFE is devoid of fatty acids, resins, waxes and coloring matters normally co-extracted by conventional solvent extraction. It also possesses an aroma more similar to the starting material from which it was derived, than an essential oil obtained by hydrodistillation or steam distillation. The almost exclusive use of compressed carbon dioxide to extract volatile concentrates or aroma substances destined to human nutrition and in the pharmaceutical and perfume industries is due to its chemical and physical properties: it is safe, non-toxic, non-combustible, inexpensive and its critical temperature and pressure are not high (31.06C and 73.82 bar) (5).

Experimental

Materials: Pimenta dioica was supplied by SardAromi (Pula, Sardinia). The plant has grown in a greenhouse and their seed came from Australia. The leaves of the plant were then air-dried in the shade for several days. For each extraction test the extractor was charged with about 200g of vegetable matter previously ground to a mean particle size of about 800 m. CO2 (purity 99%) was supplied by SIO (Societ Italiana Ossigeno, Cagliari, Italy).

SFE apparatus: Supercritical CO2 extractions were performed in a laboratory apparatus equipped with a 400 cm^sup 3^ extraction vessel, which operated in a single-pass mode by passing CO2 through the fixed bed of vegetable particles. Two fractions of the extract were recovered in two separator vessels (300 and 200 cm^sup 3^) which were connected in series. The cooling of the first separator was achieved by using a thermostatic bath (Neslab, Model CC-100II, accuracy of 0.1C). The use of the second separator allowed the discharge of the liquid product at desired time intervals. In this section, the temperature was maintained at the desired value using two methods. First by the utilisation of a heating ribbon wrapped around the piping dividing the two separators, and secondly, by means of a water thermostatic system connected to the second separator. A high pressure diaphragm pump (Lewa, Model EL 1) with a maximum capacity of 6 kg/h, pumped liquid CO2 at the desired flow rate. The CO2 was then heated to the extraction temperature in a thermostatic oven (accurate to 0.02C). Extraction was carried out in a semi-batch mode: batch charging of vegetable matter and continuous flow solvent. The flow of CO2 was monitored by a calibrated rotameter (Sho-rate, Model 1355) positioned after the last separator. The total CO2 delivered during an extraction was measured by a dry test meter. Temperatures and pressures along the extraction apparatus were measured by diermocouple and Bourdon-tube test gauges, respectively. Pressure was regulated by high pressure valves under manual control, located on different points of the apparatus.

The volatile concentrate of pimenta dioica leaves was obtained at the following conditions: P = 90 bar, T = 50C and [straight phi]^sub CO 2^ = 1-4 kg/h. The total volatiles yield, after an extraction lasting 4h, was 1.0%.

Hydrodistillation: Hydrodistillation was performed in a circulatory Clevenger-type apparatus, for 4 h, up to the point where the oil contained in the matrix was exhausted. About 100g of material belonging to the same batch employed in SFE were charged.

GC/MS analysis: A Hewlett-Packard 5890 Series II gas chromatograph, GC, was used for analysis of the extracts. It was equipped with a split-splitless injector and a DBS-MS fused silica column of 5% phenyl-methylpolysiloxane, 30 m x 0.25 mm, film thickness 0.25 m. The GC conditions used were: programmed heating from 60-280C at 3C/min, followed by 30 min under isothermal conditions. The injector was maintained at 250C. Helium, at 1.0 mL/ min, was the GC carrier gas; the sample (1 L) was injected in split mode (1:20). The GC was fitted with a quadruple mass spectrometer, MS, Model HP 5989 A. MS conditions were as follows: ionization energy 70 eV; electronic impact ion source temperature, 200C; quadruple temperature, 100C; scan rate, 1.6 scan/s; and mass range 40-500 amu. Software to handle mass spectra and to record chromatogram was MS ChemStation (Hewlett-Packard) using NIST98, and LIBR(TP) (6) mass spectra libraries. Run samples were diluted in chloroform at a dilution ratio of 1:100 (w/w). Chromatographic results were expressed as area-percentages, calculated without applying any response factor, and are reported as a function of retention indices. Identifications were made by matching both their mass spectra and retention indices, with those reported in the literature and those of pure compounds, whenever possible.

Results and Discussion

The Pimenta dioica volatile concentrate produced by SFE had a yellow color. The main constituents of the pimenta SFE (Table I) were: eugenol (77.9%), β-caryophyllene (5.1%), squalene (4.1%) and α-humulene (2.3%). The GC/MS analysis has allowed us to identify with squalene the compound at retention time of 66.7 min by matching its mass spectra and retention time values with these of pure compounds. The volatile concentrate has been compared with the oil obtained by hydrodistillation and the main differences regard the abundance of eugenol, 77.9% versus 45.4%. Indeed, the solubility of eugenol in water is not negligible; Miller and Hawthorne (7) found at 298 K the value, expressed as mole fraction of eugenol, of 1.9 10^sup -4^. So, eugenol is preferentially dissolved in the water (at 298 K, it is possible to solubilize about 1.7 g of eugenol in 1 kg of water) and therefore not fully recovered in the oil. This phenomenon determines the production of an oil which components are not in the original natural proportion and as consequence, with an altered aroma with respect to the starting material. In such case the recovery of water soluble essential oil components (8) can be necessary to improve the performance of the hydrodistillation.

Our results have been compared with those reported by Pino et al. (9) and by Garcia-Fajardo et al. (10). The difference in qualitative and quantitative analysis between our results and literature may depend on ambient and climatic conditions and different vegetative stages. Plant material used by Pino et al. was colleted from Havana; their yield, obtained during 6 hours of extraction, was 0.8% and the extract was dominated by eugenol (93.4%). In the volatile concentrate obtained by Garcia-Fajardo et al. from Mexican pimento berries the main constitent was methyl eugenol and eugenol was only 14.9%.

From the data in our possession we can conclude that, in spite of the Mediterranean climate, in which the Pimenta has grown, we have obtained a yield higher than expectations and a good quality volatile concentrate, considering the high eugenol content.

Acknowled\gements

The authors thank Pier Giuseppe Rossi (owner of SardAromi S.p.A Company) for his collaboration and for the supply of vegetable matter.

Table I. Comparative percentage composition of an SFE and oil of Pimenta dioica leaves

References

1. A. Hassanali and W. Lwande, Antipest secondary metabolites from African plants. Insecticides of Plant Origin, 78-94 (1989).

2. J.A. Klocke, M.F. Balandrin, M.A. Barnaby and R.B. Yamasaki, Limonoids, phenolics and furanocumarins as insect antifeedant, repellents and growth inhibitory compounds. Insecticides of Plant Origin, 136-149 (1989).

3. H.A.Brown, D.A. Minott, C.W. Ingram and L.A.D. Williams, Biologicalactivities of the extracts and constituents of Pimento, Pimenta dioica L. agaist the Southern Cattle Tick, Boophilus microplus. Insect Sci. Applic., 18, 9-16 (1998).

4. E. Stahl and D. Gerard, Solubility behaviour and fractionation of essential oils in dense carbon dioxide. Perfum. Flavor., 10(2), 29-37 (1985).

5. M.DeReuck, V.V. Altunin, O.G. Gadeskii, G.A. Chapela and J.S. Rowlinson, International Thermodynamic Tables of the Fluid State, Carbon dioxide. Pergamon Press, Oxford (1983).

6. R.P. Adams, Identifications of Essential Oils by Ion Trap Mass Spetroscopy. Academic Press, New York (1989).

7. D.J. Miller and S.B. Hawthorne, Solubility of liquid organic flavour and fragrance compounds in subcritical (hot/liquid) water from 298 K and 473 K. J. Chem. Eng. Data, 45, 315-318 (2000).

8. P.M.Bohra, A.S.Vaze and V.G. Pangarkar, Adsorptive recovery of water soluble essential oil components. J. Chem. Tech. Biotechnol., 60, 97-102 (1994).

9. J.A. Pino, J. Garcia and M. A. Martinez, Solvent extraction and supercritical carbon dioxide extraction of Pimenta dioica Merrill. Leaf. J. Essent. Oil Res., 9, 689-691 (1997).

10. J. Garcia-Fajardo, M. Martinez-Sosa. M. Estarron-Espinosa, G. Vilarem, A. Gaset and J. De Santos, Comparative study of the oil and supercritical CO2 extract of mexican pimento (Pimenta dioica Merrill). J. Essent. Oil Res., 9, 181-185(1997).

Bruno Marongiu,* Alessandra Piras and Silvia Porcedda

Dipartimento di Scienze Chimiche, Universit degli Studi di Cagliari, Cittadella Universitaria, St. Prov. Monserrato Sestu Km 0.770, I-09042 Monserrato, Italy

Rita Casu and Paola Pierucci

SardAromi SpA, Laboratorio di Ricerca Agrosperimentale e Chimico Estrattivo, Pula, Cagliari, Italy

* Address for correspondence

Received: May 2003

Revised: July 2003

Accepted: August 2003

1041-2905/05/0005-0530$6.00/0- 2005 Allured Publishing Corp.

Copyright Allured Publishing Corporation Sep/Oct 2005

Source: Journal of Essential Oil Research : JEOR

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