J Sci Food Agric 1993, 61, 375-376

A Simple Method for the Extraction of the Isocoumarin 6-Methoxymellein from Carrot Tissues Julien Mercier and Joseph Arul" Departement de Sciences et Technologie des Aliments, Universite Laval, Sainte-Foy, Quebec, Canada G 1 K 7P4

(Received 20 April 1992; accepted 30 November 1992)

Abstract: The use of boiling water for the extraction of 6-methoxymellein, a phytoalexin of carrot, was investigated instead of the conventional organic solvent method. The recovery (1 18 %) obtained by a two-step extraction with boiling water compared favourably with that obtained by the conventional two-step extraction with acetone.

Key words: carrot, isocoumarin, phytoalexin, 6-methoxymellein.

The isocoumarin 6-methoxymellein (6-MM) is an im- portant phytoalexin in carrot implicated in its disease resistance and is also a common stress metabolite. In studying of the elicitation of 6-MM, the time-consuming and expensive extraction and purification procedure (Kurosaki et a1 1986) for its assay in tissues is a constraint when a large number of samples are involved. While investigating the effect of food preparation on the 6-MM content of carrot, it was observed that the compound was not degraded by water boiling temperature but was leached out into the immersion water (Mercier et a1 1993b). It was then hypothesised that 6-MM could be extracted from carrot tissues by boiling water instead of the usual procedure involving extraction with organic solvents.

The accumulation of 6-MM in carrot slices was induced by exposure to ultraviolet radiation (0.22 M erg cm-2) as described by Mercier er a1 (1993a). After 6-MM was accumulated, slices were cut in half to make two identical subsamples of - 10 g. One subsample was extracted by the usual method, which involves extraction with acetone twice and partitioning according to Kurosaki et a1 (1986), but dichloromethane was used for the second partition instead of hexane. The other subsample was extracted four times by boiling for periods of 10 min in 250 ml of distilled water each. After cooling, each sample of water was partitioned with

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dichloromethane and the extracts were dried on sodium sulphate. For all extracts, 6-MM was purified by thin- layer chromatography (TLC) (benzene methanol, 100 : 2) and assayed by absorbance at 268 nm (Kurosaki et al 1986).

Boiling of carrot slices in water for 10 min followed by a partition with dichloromethane after cooling extracted larger amounts of 6-MM than the direct extraction with acetone (Table 1). The amount of 6-MM obtained by a two-step extraction with boiling water was 18 YO higher than that with acetone. This can be a result of many factors. First, the solvent capacity and selectivity of water is altered near its boiling point as evidenced by a significant decrease in its dielectric constant. At 25°C the operation of non-chemical or physical forces is very low and water behaves essentially as a protic solvent. As the temperature is raised, hydrogen bonds break causing the destruction of water's three-dimensional geometry, with concomitant increase in the population of monomeric long-range dipoles, i.e. internal pressure and non- chemical or physical force contribution to the inter- molecular bonding energies of water increase with increasing temperature (Dack 1975). Thus, the effective operation of the physical force, in particular, dipolar interactions manifest with increasing temperature which facilitates the solubilisation of substances of low polarity in water. Secondly, the release of 6-MM is promoted by softening and disruption of the tissues by heat. Finally, the loss of the substance from the manipulation should

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316 J Mercier, J Arul

TABLE 1 Effect of extraction method on the amount of 6-methoxymellein obtained from carrot slices (,ug g-I fresh weight of subsample)

Sample Standard Boiling water extraction Recot:ery solvent ratio

extraction Step 1 2 3 4 Total waterlsolvent

1 33.06 39.37 3.27 2 25.58 28.62 1.14 3 35.86 37.46 1.20

Mean 31.50 35.15 1.87 Standard deviation 5.3 1 5.74 1.21

be less as fewer number of steps are required in this procedure than the normal solvent extraction protocol. The recuperation of the residual 6-MM in the tissue by additional steps of extractions improved the extract- ability only marginally. Nevertheless, two extraction steps are desirable as it would ensure a more complete extraction of the compound.

In addition to efficiency and reduced consumption of solvents required for extraction, the extracts were also considerably purer as pigments usually extracted by organic solvents were not dissolved by hot water. Nonetheless, the extracts had to be purified by TLC to obtain the typical UV absorption spectrum of 6-MM, although this procedure was facilitated by the lower levels of contaminants. The extraction of 6-MM from carrot tissues by boiling water would thus be an efficient and less expensive alternative to the usual method employing solvents for extraction. The flexible solvent properties of water can be explored in the extraction of natural compounds as laboratories face problems of

1.15 0.63 4442 1.34 1.47 0.78 32.01 1.25 1.38 0.79 40.83 1.14 1.33 0.73 39.09 1.24 0.17 0.09 6.39 0.10

disposal of organic solvents and consider alternative methods such as supercritical fluid extraction (France et al 1991).

REFERENCES

Dack M R J 1975 The importance of solvent internal pressure and cohesion of solution phenomena. Chem Soc Rev 4

France J E, King J W, Snyder J N 1991. Supercritical fluid- based cleanup technique for the separation of organochlorine pesticides from fats. J Agric Food Chem 39 1871-1874.

Kurosaki F, Amin M, Nishi A 1986 Induction of phytoalexin production and accumulation of phenolic compounds in cultured carrot cells. Physiol Mol Plant Pathol 28 359-370.

Mercier J, Arul J, Ponnampalam R, Boulet M 1993a Induction of 6-methoxymellein and resistance to storage pathogens in carrot slices by UV-C. J Phytopathol (in press).

Mercier J, Arul J, Julien C 1993b Effect of food preparation on the content of the isocoumarin 6-methoxymellein in UV- treated carrots. Food Res Intern (submitted).

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