Abstract

PIGMENTS AND THEIR SOLUBILITY IN AND EXTRACTABILITY BY SUPERCRITICAL CO₂ - I: THE CASE OF CURCUMIN

W. Baumann¹, S. V. Rodrigues² and L. M. Viana3

1Institut für Physikalische Chemie, Universität Mainz, 55099 Mainz, Germany

² Departamento de Química Analítica, Universidade Federal Fluminense,

Outeiro de São João Batista s/n, 24020-150, Niterói - RJ, Brazil,

³ Departamento de Química Orgânica, Universidade Federal Fluminense,

Outeiro de São João Batista s/n, 24020-150, Niterói - RJ, Brazil

(Receiveid: February 10, 2000 ; Accepted: May 5, 2000)

INTRODUCTION

Determination of the solubilities of some triazine pesticides in supercritical (sc) CO₂ using the saturated absorption of the resulting solution was demonstrated using a high-pressure photometric cell, specially designed to fit into a Perkin Elmer spectrophotometer Lambda 5. The design of this cell involves a magnetic stirrer and has an optical pass adjustable from 0 to 6 mm (Rodrigues et al., 1998). In the case of pigments, not only their solubility but also their extractability from plant matrices can be determined if the desired pigment has a sufficiently spectrally isolated visible or UV absorption band, not superimposed by any other compound present in the extracted matrix. This will be demonstrated in the present communication for the extraction of curcumin (1,7-Bis(4-hydroxy-3-methoxyphenyl)-1,6-heptadiene-3,5-dione) from turmeric, which is the ground dried rhizome of curcuma longa L. In fact, extraction yields what is called oleoresin (see Govindarajan, 1980 and Sanagi et al., 1993) and which contains as colorants dominantly curcumin and in minor quantities the desmethoxy and the bisdesmethoxy derivatives of curcumin. This is what is used as a spice and a colorant, and it is this mixture which is - not quite precisely - often called curcumin. In this publication we shall quantify curcumin relative to the curcumin standard from Merck, by comparing the UV-absorbance of the standard and the extract. We shall not refer to medical applications of curcumin as an anti-inflammatory antioxidant (e.g., Masuda, 1997) or to its use in the determination of boron (e.g., Sah and Brown,1997).

EXPERIMENTAL

Apparatus

The experimental setup is described in Rodrigues et al., 1998, and was mounted in the laboratory of the Department of Analytical Chemistry at the Universidade Federal Fluminense in Niterói and also, with minor modifications, at the Institute of Physical Chemistry of the University of Mainz (see Fig. 1). It consists mainly of a Perkin Elmer Lambda 15 spectral photometer (Niterói), used at 2 nm bandwidth, or of a Perkin Elmer model Lambda 5 (Mainz), especially equipped with a fixed 1 nm optical bandwidth, and a membrane compressor model 554 2121 from Nova Swiss with a high-pressure regulating valve model HPR 802-S-4 from Veriflow. In both laboratories the same high-pressure photometric cell, of our own design and construction (volume = 1.7 ml), was used, the optical path adjusted to (well-defined) 5 to 6 mm with the extractability experiments and with the solubility experiments in pure CO₂, but to 0.5 mm for the solubility experiments in modified CO₂.

CO₂ was supercritical fluid chromatography quality from White Martins, Rio de Janeiro, or 5.5 quality from Messer-Griesheim, Frankfurt, both with sufficiently low optical density down to 190 nm. Ethanol (96%) used as modifier was spectroscopic grade, especially treated for high UV transmittance in WB´s laboratory.

The curcumin reference standard R was from Merck-Schuchardt, product number 820354 (lot n° 53516062). It is specified by acidimetric analysis for 97% curcumin content and characterized by its melting point at 170 - 175°C. It was used without further purification.

Turmeric samples were the following spice samples: SB, "Kurkumin" from Brecht, Germany; SS, "Curcuma" from Spice Islands, lot n° LO 56; and SV dried and coarsely milled root of curcuma longa L. from AINIA, Valencia/Spain, harvested in 1993 at an unknown location in Asia.

Determination of extractability

Step 1 A known small amount m_R of the pure reference compound R under consideration is completely dissolved in the high-pressure cell under the desired pressure/temperature conditions. After equilibrium has been reached the asymptotic absorption A_R in the cell is measured (of course, the baseline is to be determined at each considered pressure value for correction, too).

Step 2 A known amount m_1S of the sample matrix S to be extracted is then put into the cell, the increasing absorption is observed and its asymptotic value A_S is measured.

The relative content C_S of R in the sample matrix S is then

C_S = 100 * m_R * A_S / A_R / m_S,

where C_S is given in mass % of the sample matrix.

Cell volume and optical path need not be determined since they cancel in this experiment!

Errors

Reference samples R were solutions of precisely weighed amounts of R in weighed amounts of methylene chloride, from which the concentration was calculated (density r = 1.324 g/ml) and a microliter syringe was used to inject a known amount m_R of the sample R into the cell, through a well-defined volume. Methylene chloride was allowed to evaporate at 47°C before the cell was closed, thermostatted to 40°C and then pressurized.

The overall statistical error from weighing, through sample injection and equilibration to absorbance readings can be estimated from the average absorption over 5 independent determinations of the absorbance in the cell at the band maximum at 405 nm, which was (0.0702 ± 0.0045) per µg substance R in the cell at (6.08 ± 0.1) mm optical path, at 40°C and 180 bar, in CO₂ modified with 10% (v/v) ethanol. Using only the three best values, this factor is 0.0704 ± 0.0013. With the molecular weight of curcumin (M = 368.37 g/mol) the absorption coefficient in the maximum at 405 nm is calculated from the latter values to be (72500±2000) l mol^-1 cm^-1, with additional calibration error of the balance and the syringes of about 5%. The absorption coefficient is 10 to 20% higher than reported in the literature (Tonnensen et al., 1995 and Jasim and Ali, 1989) for different (liquid) solvents.

Turmeric samples SS, SB and SV were weighed on a sheet of Nickel, by subtracting the sheet weight after transferring the sample to the cell from the total sample plus sheet weight, so that incomplete transfer to the cell would not cause error. Amounts of substance were between 0.5 and 1.6 mg. The main error with the extractability experiments stems from the slow extraction kinetics, especially slow with dry CO₂ - the time to reach the asymptotic absorption was up to 1^1/2 hours with fine milled samples of the order of 1 mg. This was caused by the special structure of the tissue of the curcuma root longa L., rather than the low solubility of curcumin in sc CO₂.

RESULTS

The method is demonstrated with curcumin from turmeric samples. Fig. 2 shows the absorption spectra of the Merck curcumin reference R in pure CO₂ and in CO₂ modified by 10 %(v/v) ethanol, both at 40°C and 240 bar. The polar modifier causes band broadening and a slight red shift, as is expected. In addition, the spectrum in pure ethanol is shown. Of course it is even more broadened and red shifted.

Fig. 3 shows the absorption spectrum of R and of SS, both at 40°C and at 180 bar. The spectra do not differ to a significant degree within the long wavelength band, but do so at short wavelengths, with a dominant band around 230 nm, with SS (and also with SB, not shown here). For the data shown in Fig. 3 the ratio of the absorptions of SS and R is 1.15 ± 0.02, from 390 to 460 nm (1.16 ± 0.05 from 390 to 470 nm). Hence, if this spectral region is used, the quantification of the curcumin content of turmeric relative to R is not disturbed by compounds interfering within the depicted spectral region.

The short wavelength band may be mainly ascribed to sesquiterpenoids and furanosesquiterpenoids which have been shown to be major constituents besides curcumin (Ma et al., 1995) in the related species curcuma zedoaria.

The results from extraction of three different turmeric samples with sc CO₂ modified by ethanol and in pure ethanol are shown in Table 1.

DISCUSSION

The results are in agreement with known curcumin contents. Differences when curcumin is extracted with ethanol modified CO₂ and with pure ethanol are within the sample inhomogeneity, although the samples were of fine milled quality (SB and SS) or were milled and mixed as thoroughly as possible (SV). After 15 days, only the sample SV showed an increased extracted content (4.1%). The other samples were extracted completely by modified CO₂. This difference in behaviour and total content of curcumin might be ascribed to the fact that the sample SV was ground freshly and not as fine as the commercial products prior to extraction, whereas SB and SS had already been aged.

It is important to note that under the static extraction conditions used here a sufficient ethanol content must be present in the extractor cell, sufficient with respect to its equilibrium distribution between the solid curcumin sample and the supercritical phase. This can be seen from an experiment, where the sample amounts were considerably increased compared to the 1 mg amounts used for the experiments of table 1. Table 2 presents these results.

50 µl ethanol in 1.7 ml cell volume have been sufficient to extract 1 mg root samples completely. With the same 50 µl in 1.4 ml cell volume (here 1.11 mm optical path) only a very small amount of curcumin could be extracted from 14 mg samples and even 170 µl of ethanol are not sufficient to extract all the curcumin. This must be ascribed to the adsorption of ethanol on the curcuma root matrix, since the equilibrium distribution of ethanol between the root matrix and the sc CO₂ phase is quite in favour of the root. This, of course, can be understood qualitatively considering the polarity of the species involved.

This hypothesis is also supported by the UV/VIS absorption spectrum of the sc CO₂ solution which in the case of the 14.2 mg sample with 50µl ethanol (Table 2) shows its maximum at 400 nm, unlike the experiments with smaller root matrix amounts, where the maximum is around 404 - 405 nm. Thus less solubilization of solute curcumin is indicated, which means less ethanol content in the supercritical phase. Furthermore, this result clearly shows that the definition of the supercritical two-component CO₂ / modifier phase by the stoichiometry used alone might not, in general, be correct in cases of extraction of compounds from plant matrices. A sufficient excess of modifier should be used under static extraction conditions.

ACKNOWLEDGEMENTS

Financial support by the German Academic Exchange Office (DAAD) and of FAPERJ is gratefully acknowledged.

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