Selectivity in the extraction of 2- quinolone alkaloids with supercritical CO2

Abstract

This work describes the conditions for selective extraction of the alkaloids in Andreadoxa flava (Kallunki). The experiments were performed in a pilot plant SFE 500, in the following conditions. Extractor pressures 206.8 x 10(5), 241.3 x 10(5), 275.8 x 10(5) and 310.3 x 10(5) Pascal and pressures of 137.9 x 10(5), 69.0 x 10(5) and 55.2 x 10(5) Pascal for separators 6, 7 and 8 respectively. The temperatures were 40 °C in the extractor and 20 ºC in the separators. The samples, extracted from A. flava leaves, were analysed by ¹H NMR and GC/FID as well as by comparison with standards. This procedure allowed the identification for the first time of the 2-quinolone alkaloid 8-methoxy-N-methyl-flindersine in A. flava leaves of the. In addition, zanthophylline, 5-methoxyalmene and 8-methoxyflindersine were identified as the main alkaloids in leaves. Under the best conditions for zanthophylline extraction it was possible to obtain a purity of 79.4% while with other phytochemical technique 55.5% was obtained.

Supercritical CO2 extraction; Alkaloids; 2-quinolone; Andreadoxa flava

THERMODYNAMICS AND SEPARATION PROCESSES

Selectivity in the extraction of 2- quinolone alkaloids with supercritical CO₂

L. L. B. Santana^I; L. A. Cardoso^II; J. I. Druzian^III; V. F. Souza ^II; T. A. C. Costa^II; D. A. Nóbrega^II; S. V. A. Hohlemwerger^I; E. S. VelozoI, ^*

^IDepartamento do Medicamento, Laboratório de Pesquisa em Matéria Médica (LAPEMM), Faculdade de Farmácia , Universidade Federal da Bahia, UFBA, Phone, Fax: +(55) (71) 3332-1580, R. Barão de Jeremoabo s/n, Campus Universitário de Ondina, CEP 40.170-290 Salvador - Bahia, Brazil. E-mail: [e-mail]

^IIDepartamento de Química Orgânica, Instituto de Química, GESNAT (AGRONEX), UFBA, CEP 40.170-290 Salvador - Bahia, Brazil

^IIIDepartamento de Análises Bromatológicas, Laboratório de Cromatografia, Faculdade de Farmácia, UFBA, CEP 40.170-290 Salvador - Bahia, Brazil

ABSTRACT

This work describes the conditions for selective extraction of the alkaloids in Andreadoxa flava (Kallunki). The experiments were performed in a pilot plant SFE 500, in the following conditions. Extractor pressures 206.8 x 10⁵, 241.3 x 10⁵, 275.8 x 10⁵ and 310.3 x 10⁵ Pascal and pressures of 137.9 x 10⁵, 69.0 x 10⁵ and 55.2 x 10⁵ Pascal for separators 6, 7 and 8 respectively. The temperatures were 40 °C in the extractor and 20 ºC in the separators. The samples, extracted from A. flava leaves, were analysed by ¹H NMR and GC/FID as well as by comparison with standards. This procedure allowed the identification for the first time of the 2-quinolone alkaloid 8-methoxy-N-methyl-flindersine in A. flava leaves of the. In addition, zanthophylline, 5-methoxyalmene and 8-methoxyflindersine were identified as the main alkaloids in leaves. Under the best conditions for zanthophylline extraction it was possible to obtain a purity of 79.4% while with other phytochemical technique 55.5% was obtained.

Keywords: Supercritical CO₂ extraction; Alkaloids; 2-quinolone; Andreadoxa flava.

INTRODUCTION

Superior plants have a large variety of micromolecules. These metabolites are used in perfumes, personal hygiene products and in the pharmaceutical and food industries. These substances can be classified in accordance with its biosynthetic origin. Alkaloids are among the most diversified classes of natural products. These substances are generally, bitter taste and have different biological properties, and some of them have a therapeutical effect on human beings when administered in appropriate doses [Simões et al., 1999]. The literature describes important therapeutical activities for 2-quinolone alkaloids such as antineoplasics, sedatives and antiarrhythmics. A previous study demonstrated that Andreadoxa flava, belonging to the Rutaceae family, is a rich source of this class alkaloid [Hohlemwerger et al., 2004].

These nitrogen containing structures can be extracted with organic solvents or by acid-base extraction, when basicity allows. In purification conventional phytochemical techniques such as recrystallization and classic chromatographic separation are used. However, these methods are limited as the require of high temperatures for elimination of the solvents, it is difficult to remove all residual solvent and the variation in pH can produce chemical alterations and possible toxic effects in the final product [Maul et al., 1998; Ndiomu and Simpson, 1998 ].

Extraction by supercritical fluid is an alternative to the conventional processes that allows to obtain high-quality products using a clean technology combined with the high efficiency of processing [Freitas et al., 2001]. Substances like caffeine, theobromine, quinine, morphine, theophilline and nicotine vary basicity and polarity and have been obtained by supercritical fluid extraction both with and without the aid of a co-solvent [Johannsen and Bruner, 1994; Lim and Hartland, 1996].

The objective of this study was to establish some parameters for the selective extraction of 2-quinolone alkaloids from Andreadoxa flava leaves using supercritical CO₂.

MATERIALS AND METHODS

Andreadoxa flava (Kallunki) leaves, collected in the city of Ilhéus (BA), were dried at room temperature, triturated and later submitted to supercritical extraction CO₂ in a SFE 500 pilot plant, which is illustrated in Figure 1. This unit is composed of a cylinder contends CO_2; a condenser (2); a pump (3) for the transport of CO₂ to the 400 mL extractor (5); after passing through a heater (4) and an outflow measurer (12); three separators (6, 7, 8) 16 mL each; a filter containing activated charcoal (1) to hold back the material that passes through the separators when the CO₂ is recycled; a cooling bath (9); and two hea baths (10 and 11).

The samples collected after extraction with supercritical CO₂ were analyzed using thin-layer chromatography (TLC) where after elution the plates of the TLC were developed with ultraviolet light at 240 nm and 360 nm, iodine and Dragendorff reagent for alkaloid detection. Some samples were analyzed by proton nuclear magnetic resonance (PNMR) in a spectrometer operating at 300 MHz in CDCl₃ and having tetramethylsilane as internal standard. The components of each collected fraction were identified and quantified using a Perkin Elmer gas chromatograph (GC) with flame ionization detector (FID) equipped with a silica column (carbowax 30 m length x 0.25 mm internal diameter and 0.25 µm of thickness film). The initial temperature of the oven was 60 °C for 5 minutes. Later the temperature was raised 10 °C for minute up to 280ºC and then maintained at this level for 20 minutes. Four samples were extracted, each one of 30 g of dried and triturated A. flava leaves. The conditions applied are described in Table 1. This procedure generated extracts A to D.

RESULTS AND DISCUSSION

All the TLC of the fractions collected under the different conditions indicated the presence of alkaloids. The samples with high alkaloids contents were selected for analysis by PNMR. The spectra of the A-3 fraction (Figure 2), obtained in the third separator the first condition of extraction, had signals identical to the ones described in the literature for the 2- quinolone alkaloid zanthophylline (I) [Hohlemwerger at al., 2004].

The TLC still made possible the detection of other alkaloids in the fractions. For identification, the fractions obtained in the supercritical fluid extraction and previously isolated alkaloid standards for A. flava were analyzed by GC/FID. The criteria of choice of the samples for analysis by GC were yield and indication of the presence of alkaloids. These were mainly the samples collected in the extractor and the third separator under each condition. A comparison of the retention times (t_R) of substances in the chromatographs of the samples obtained with the times of retention of the 2-quinolone alkaloid standards made it possible to identify (I) zanthophylline, t_R = 25,86 min; (II) 8-methoxyflindersine, t_R = 25,25 min; (III) 5-methoxyalmene, t_R = 24,86 min and (IV) 8-methoxy-N-methyl-flindersine, t_R = 24,19 min, with the last one being identified for the first time in A. flava leaves. In Figures 3 and 4 the structures of these substances and the chromatograph of the A-9 fraction, respectively, are shown.

Besides identification of the alkaloids, it was also possible to calculate the percentage of each component in the selected samples, using the normalization method. These values are presented in Table 2.

A comparison of the compositions of samples obtained under the conditions tested allowed verification that the zanthophylline alkaloid, because of its high solubility in CO₂, is present in the majority of the samples. Fractions A-3, A-9, B-4, C-4 and D-4, collected in the third separator, showed the largest amounts of this substance. The fraction collected in the third separator at the highest extraction pressure (310.3 X 10⁵ Pa) had the largest amount of zanthophylline. The results indicate that the density of CO₂ is an important factor in the selectivity of the extraction for this alkaloids. The present work also compared SCFE with the conventional phytochemical techniques for attainment of these alkaloids. The chromatograph presented in Figures 5 and 6 provide evidence of the selectivity of SCFE compared to extraction using conventional phytochemical methods.

The conditions used for SCFE allowed the attainment of zanthophylline 79,4% purity, while with the use of conventional phytochemical methods this alkaloid was obtained at a purity of 55.5% at most.

CONCLUSIONS

This study enabled the identification of 8-methoxy-N-methyl-flindersine alkaloid in A. flava leaves, not detected by traditional phytochemical methods. It also established a method for the selective extraction of 2-quinolone alkaloids, mainly the zanthophylline alkaloid from A. flava leaves, proving that selectivity of extraction with supercritical CO₂ is greater than that with the conventional extraction methods. The fraction collected in the third separator at the highest extraction pressure (310.3 X 10⁵ Pa) had the largest amount of Zanthophylline. The results indicate that the density of CO² is an important fator of selectivity in the extraction of this class of alkaloids.

Received: October 20, 2004

Accepted: May 10, 2006

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*

To whom correspondence should be addressed

Publication Dates

• Publication in this collection
  25 Apr 2007
• Date of issue
  Dec 2006

History

• Accepted
  10 May 2006
• Received
  20 Oct 2004