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Melissa officinalis, L.: study of antioxidant activity in supercritical residues
Journal of Supercritical Fluids 21 (2001) 51 – 60 www.elsevier.com/locate/supflu
Melissa officinalis, L.: study of antioxidant activity in supercritical residues M.A. Ribeiro *, M.G. Bernardo-Gil, M.M. Esquı´vel Centro de Engenharia Biolo´gica e Quimica, Departamento de Engenharia Quı´mica, Instituto Superior Te´cnico, A6. Ro6isco Pais, 1049 -001 Lisboa, Portugal Received 4 May 2000; received in revised form 1 March 2001; accepted 13 March 2001
Abstract The supercritical CO2 extraction of lemon balm (Melissa officinalis, L.) at pressures from 10 to 18 MPa and at temperatures of 308–313 K was studied. The antioxidant activity of lemon balm extracts, obtained from solid residues of supercritical extraction and from raw lemon balm leaves, was performed using the Rancimat method. The best protection factor curve was obtained when extracts from the solid residues of supercritical extraction at 10 MPa, 308 K and 4 h of extraction time were used. A spectrophotometric method was used for the determination of the polyphenol compounds in the extraction residues. The highest value of phenol compounds was obtained for the extracts of solid residues of supercritical extraction at 10 MPa, 323 K and 30 min. © 2001 Elsevier Science B.V. All rights reserved. Keywords: Antioxidant; Lemon balm; Polyphenol; Rancimat; Supercritical extraction
1. Introduction Lemon balm (Melissa officinalis, L.) is known as an officinal herb of a long tradition and a large variety of uses. Many of the therapeutic effects of this herb are attributed to its leaf essential oil, which is rich in aldehydes and terpenic alcohols. The main constituents are citral (geranial and neral), citronellal, linalool, geraniol, b-caryophyllene and b-caryophyllene oxide, comprising about 96% of the oil ingredients [1]. Therefore, it is not * Corresponding author. Tel.: + 351-21-8417312; Fax: + 351-21-8499246. E-mail address: [email protected] (M.A. Ribeiro).
surprising that lemon balm oil has a special reputation among essential oils. As a result of the low content of essential oil (balm leaves normally contain 0.1% approximately), balm oil has a very high price level [2]. The differences encountered between the oils obtained from different origin countries from dried whole plants or dried leaves are in the yield, rather than in the qualitative composition [3]. Generally, the antioxidant effect has been explained by the two following mechanisms. One mechanism is ascribed to scavenging free radicals such as peroxy and alkyl radicals to break the chain reaction. Another one is ascribed to the decomposition of hydroperoxides [4].
0896-8446/01/$ - see front matter © 2001 Elsevier Science B.V. All rights reserved. PII: S 0 8 9 6 - 8 4 4 6 ( 0 1 ) 0 0 0 7 8 - X
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M.A. Ribeiro et al. / J. of Supercritical Fluids 21 (2001) 51–60
The oxidation of unsaturated lipids has been extensively studied since it relates to deterioration of muscle foods, production of both desirable and undesirable breakdown products, and numerous reactions associated with other food constituents [5]. Synthetic phenolic compounds, such as butylated hydroxyanisole (BHA), butylated hydroxytoluene (BHT) and tert-butylhydroquinone (TBHQ), have been widely used as antioxidants in food lipids. However, possible toxicity and/or mutagenecity of these antioxidants have been a subject of study for many years. There are several recent reports on the effect of BHA on conversion of ingested material into toxic substances or carcinogens due to increase secretion of microsomal enzymes of liver and extra hepatic organs, such as the lungs and gastrointestinal tract mucosa. Therefore, at the present time, the Food and Drug Administration in the US is examining possible removal of BHA from the Generally Recognised As Safe list [6]. Furthermore, having been reported that BHT is carcinogenic in rats, this antioxidant is also in the process of being carefully scrutinised. In addition, TBHQ has not been approved for food use in Europe, Japan and Canada. Thus, natural antioxidants have gained popularity in recent years [6]. Chlorophyll is an important photosensitizer in plant-derived foods, although the presence of carotenoids in the chloroplast normally yields protection by quenching of singlet oxygen. This protection may, however, be impaired by drying and extraction of herbs. Exposures to light change this antioxidative effect to a pro-oxidative effect [7]. For storage in light, a relatively poor antioxidative effect has been seen for plants with higher quantities of chlorophyll. Naturally occurring antioxidative components in foods include flavonoids, phenolic acids, lignan percursors, terpenes, mixed tocopherols, phospholipids, polyfunctional organic acids and also plant extracts. The compounds in lemon balm (M. officinalis) that showed antioxidant activity, caffeic acid and rosmarinic acid, were more active than a-tocopherol, having an activity comparable with the BHA.
Rosmarinic acid (a-o-caffeoyl-3,4-dihydroxyphenallatic acid) is one of the most abundant caffeic acid esters occurring in plants. It is mainly found in species of the Boraginaceae and Lamiaceae families, and is noted for its potent antioxidant properties [8]. Current research on rosmarinic acid centres on its physiological and pharmacological activities. Oxidised rosmarinic acid has displayed antithyrotropic activity in testes with human thyroid membrane preparations, and the pure compound has been shown to effectively suppress the complement-dependent components of endo toxin shock in rabbits. Rosmarinic acid is also known to react rapidly with viral coat proteins and so inactivate the virus. There seems to be sufficient promise as regards its useful properties to have led at least one pharmaceutical firm to undertake a serious examination of rosmarinic acid as a potential pharmaceutical plant product [8]. The majority of extractions carried out for the food, flavouring and perfumery industries will be on solid vegetable starting materials. Plant materials usually have a low bulk density (typically 500 kg m − 3) and a fairly low extractable content, which obliges the use of large volume extractors. Supercritical fluid technology is often considered expensive due to very high investment costs in comparison with classical low-pressure equipment. Most companies believe that it leads to high-quality products, but it is in fact restricted to high-added value products. However, this is far from true when very large volumes of materials are treated [9]. Carbon dioxide is a very non-polar solvent with characteristics similar to pentane or hexane. Commercial CO2 is obtained as a by-product of fermentation processes, so its use as an extraction solvent does not increase the amount already present in the atmosphere. Therefore, there is no overall detrimental effect on the earth’s ozone layer from this use of CO2 [10]. Although extensive R&D investigations have been carried out world wide for more than 25 years, it is disappointing that supercritical fluid applications have been still limited to few areas; and it does not appear that development will rocket in the near future. However, this should
M.A. Ribeiro et al. / J. of Supercritical Fluids 21 (2001) 51–60
not lead one to give up the numerous opportunities arising now, from food ingredients and nutraceuticals to pharmaceuticals, from biological applications and pollution abatement to new material manufacture [9]. The aim of this work was to obtain extracts with antioxidant power. Two solid materials were studied: 1. lemon balm leaves; and 2. the solid residues obtained after the supercritical extraction of the oil. Parameters such as pressure, temperature and extraction time were studied. The pressure of the first collector was also analysed. The influence of theses parameters on the antioxidant activity of the extracts (ASR) was performed using the Rancimat method. Determination of the polyphenols compounds in the extraction residue was carried out using a spectrophotometric method.
2. Materials and methods Lemon balm was collected in Portugal and was used as received (air-dried and coarsely cut). The CO2 used in this work was 99.5% pure (w/w), and was supplied by Ar Liquido (Portugal). The standard Rosmarinic acid was purchased from Extrasynthese (Genay, France). All the other chemicals used in this work were obtained from various commercial suppliers and were of the highest purity available.
2.1. Supercritical extraction The main interest of the supercritical extraction experiments was on the solid residue of the extraction, to be used as raw material for antioxidant extraction. Extraction measurements were carried out in a semi-batch flow extraction apparatus. The supercritical extraction apparatus has been described elsewhere [11]. It mainly consisted of a 500 ml extractor (L/D =5.8), two 200 ml separation vessels type cyclones SFE500 (Separex, Champigneulles, France), operated in series and operating at different pressures and temperatures, called in this
53
work the first and second collectors. A glass coil (immersed in a dry ice/acetone bath) was placed at the exit of the apparatus at about 203 K and at ambient pressure. The volume of carbon dioxide delivered was measured by a Dry Test Metter (American Meter Company, Philadelphia, USA).
2.2. Antioxidant extraction 2.2.1. Preparation of lemon balm extracts The ground lemon balm leaves and ground supercritical extraction solid residues (Moulinex coffee grinder) were boiled with distilled water for about 1.5 h, using an agitation plate and a magnetic stirrer. The homogenate was then filtered through a coffee filter paper; the collected filtrate was acidulated with a solution of 25% HCl to pH 2.5 to precipitate the waxes. After filtration with a Whatman number 1 filter paper, the aqueous phase was mixed with diisopropilether (10:3) and allowed to separate. The upper aqueous layer was extracted twice. The organic phases were combined and dryness with MgSO4, anhydrous filtered and then evaporated using a rotary evaporator (Heidolph VV2000). Schemes of the main operations for the preparation of lemon balm antioxidant from supercritical residues (ASR) and from raw lemon balm leaves (ARM) are shown in Figs. 1 and 2, respectively. 2.3. Polyphenol determination A quantitative determination of total phenols by a spectrophotometric method has been carried out using a spectrophotometer (Unicam Helios a). The antioxidant extracted with diisopropilether was dissolved in a mixture of methanol:water (6:4) to obtain a solution of 100 p.p.m. in antioxidant extract. On a volumetric flask of 25 ml has been put 17.5 ml distilled water, 1 ml solution methanol:water (6:4) and 1.25 ml Folin reagent (mixture of phosphomolibdic and phosphowolframic acid). The solution was agitated and rested for 3 min, after which was added 2.5 ml Na2CO3
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M.A. Ribeiro et al. / J. of Supercritical Fluids 21 (2001) 51–60
solution (20%), and the volume of the flask completed with distilled water.
The absorbance at 750 nm was measured after 1 h in a dark camera. The spectrophotometric calibration curve with different concentrations of standard rosmarinic acid has been carried out. The white and the standard solutions were prepared in the same way but without added polyphenol solution (in the first case) and with an added 1 ml rosmarinic acid standard solution with a concentration of 50 p.p.m. (in the second case) [12]. The results were expressed in rosmarinic acid because it is one of the major compounds present in lemon balm, and all the extractions and purification were carried out to obtain a purified rosmarinic acid.
2.4. Rancimat method
Fig. 1. Scheme for the preparation of antioxidant extracts from supercritical residues of lemon balm (ASR).
The rancimat method (Metrohm Rancimat 679) was used for the determination of the antioxidant activity. Samples of extracts dissolved in sunflower oil at a concentration range from 200 to 4000 p.p.m. were heated at 393 K. A continuous air stream (20 l h − 1) at ambient condition was passed through the heated samples and the volatile compounds were absorbed in a conductivity cell. The conductivity was monitored continuously until a sudden rise signified the end of the induction period [11].
3. Results and discussion
Fig. 2. Scheme for the preparation of antioxidant extracts from raw material (ARM).
Antioxidant extract yield from raw material, lemon balm leaves (ARM) was 0.4 g extract/100 g plant material. When the ground supercritical solid residues (ASR) were used, yields of 0.6 g extract/100 g supercritical solid residue were obtained, independently of the extraction pressure. The interest of this work was not the study of essential oil extraction or the study of its fractionation, although it was observed that the pressure in the first collector was of primordial importance for the total mass of extract recovered. As can be seen in Fig. 3, for the same pressure and temperature of the extraction, respectively, of 12 MPa and 313 K, and a superficial velocity of carbon dioxide of 0.02 cm s − 1 but different pres-
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Fig. 3. Percentage of solute recovered for a extraction condition of 12 MPa, 313 K, a superficial velocity of carbon dioxide of 0.02 cms − 1 and different pressures of the first collector: , 8–9 MPa; , 6 – 7MPa.
sure of the first collector, the amount of extract collected was quite different. High recoveries (30%) were found when the pressure of the first collector was 8–9 MPa, but only 8% (w/w) were obtained when the pressure of the first collector varies between 6 and 7 MPa. The percentage of solute collected was calculated by dividing the total mass of solute collected by the weight of material charged in the extractor. In the first collector, the cuticular waxes were selectively precipitated, which is normal because the alkanes are extracted more rapidly with pure CO2 than the essential oil components, which might be expected since plant waxes are found on the tissue surface. However, for pressures under 8 MPa, the high molecular weight compounds could not be recovered because of the poor collection efficiencies, which result in low recoveries and could mistakenly be blamed on poor extraction efficiencies [13].
The spectophotometric polyphenol determination was expressed as the percentage of rosmarinic acid in antioxidant extracts of M. officinalis, because it is one of the major compounds present in lemon balm, and the experiments were carried out to perform a purification of rosmarinic acid. This concentration depends on the extraction conditions used, varying between 10 and 80%, as presented in Fig. 4. The highest concentration of phenol compounds was observed for the solid residues obtained at 10 MPa, 323 K and 0.5 h of extraction. For this extraction condition, the oily extract was green, having a great quantity of chlorophyll, which was confirmed spectrophotometrically at 600 nm. If great quantities of chlorophyll were extracted, the remaining amount of unwanted compounds that can minimize or hide the polyphenol determination in solid residues was
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lower and, subsequently, the phenol percentage was higher. The protection factor (PF) was calculated by dividing the induction time of the sample by the induction time of sunflower oil. When materials do not have antioxidant activity, the induction time of their dispersion is equal to the induction time of the control sample (sunflower oil), and PF is equal to 1. A PF greater than 1 indicates antioxidant activity, while a PF less than 1 indicates pro-oxidant activity. In Figs. 5–7, the protection factors of extracts were plotted as a function of extract concentration (ASR) in sunflower oil at different pressures but at constant extraction times of 0.5, 4, and 20 h, respectively. For a short extraction time of 0.5
h, the antioxidant activity of the ASR extracts was not greatly affected by the pressure and temperature, but for higher extraction times the behaviour of the sample was different. These two different trends seem to be justified taking into account that, for the shorter extraction time 0.5 h, the only extractable compounds were volatile compounds and cuticular waxes, located on the surface of the vegetable matter, and the extraction conditions did not seem to interfere significantly with the extraction of components that influence the antioxidant activity of the supercritical solid residues (ASR). But this was not true for the high extraction time of 4 h, where the balance between solvent power and selectivity must be pondered. High densities induce high
Fig. 4. Percentage of phenol on extracts of supercritical solid residues (ASR), and for the extract of M. officinalis (ARM).
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Fig. 5. Protection factor versus concentration of antioxidant extracts (ASR) for an extraction time of 0.5 h, a superficial velocity of carbon dioxide of 0.02 cm s − 1, and different conditions of pressure and temperature: , 18 MPa, 323 K; *, 10 MPa, 308 K; , 10 MPa, 323 K.
solvent power and low selectivity. The balance between these two important factors, solvent power and selectivity of the supercritical CO2 is perhaps the most important role to obtain a supercritical solid residue with higher antioxidant activity. Natural materials contain various extractable materials. Among these, the waxes greatly hamper the preparation of antioxidant extracts because the filtration of the water solution (ground leaves boiled with distillate water) was quite difficult. Without these types of compounds, the filtration time will be much improved. It was possible to spare heaps of time. For the long extraction time of 20 h, as can be seen in Fig. 7, the antioxidant activity was lower than for shorter extraction time (Figs. 5 and 6) at the same concentration of solution. For long extraction times, compounds with great chains like flavonoids, flavonoics, triterpe-
nes, and organic acids began to be extracted. These classes of compounds typical of oleoresins show a very stable antioxidant activity when in synergy with phenol compounds. If these compounds are extracted, its concentration in the residue is lower and, subsequently, the stability of the antioxidant activity is affected. This is the reason why, at the same concentration, extracts obtained from long extraction times show lower antioxidant activity. Besides, if the extraction pressure increase for the same conditions of temperature and velocity of carbon dioxide, the solvent power increase and, consequently, the antioxidant activity of the residue decrease, because as has already been said the compounds with great chains began to be extracted. On a closer view of the Fig. 8, the antioxidant activity of extracts obtained from supercritical extraction residues of lemon balm increased; all of
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them with increasing concentrations of the extracts. But, as has been previously discussed, the behaviour of the extracts changed at an aleatory form,
independent of the extraction conditions used. The best curve was obtained for the condition of 10 MPa, 308 K and 4 h of extraction.
Fig. 6. Protection factor versus concentration of antioxidant extract (ASR) for an extraction time of 4 h, a superficial velocity of carbon dioxide of 0.02 cm s − 1, and different conditions of pressure and temperature: *, 10 MPa, 308 K; , 10 MPa, 323 K; , 18 MPa, 323 K.
Fig. 7. Protection factor versus concentration of antioxidant extract (ASR) in sunflower oil for long extraction time, 20 h, and temperature of 313 K and a superficial velocity of CO2 of 0.02 cm s − 1: ", 12 MPa; ×, 18 MPa.
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Fig. 8. Protection factor versus concentration of antioxidant extract in sunflower oil: , extracted lemon balm (ARM); —, BHT; *, 10 MPa, 308 K, 4 h; , 18 MPa, 323 K, 0.5 h; , 12 MPa, 308 K, 2 h; ", 12 MPa, 313 K, 20 h; ×, 18 MPa, 313 K, 20 h;
, 10 MPa, 323 K, 0.5 h; , Tocobiol; , 10 MPa, 308 K, 0.5 h; , BHA; +, 18 MPa, 323 K, 4 h.
4. Conclusions The best pressures for the first collector, if we were not interested in essential oil and subsequently its fractionation, but only in the residue, are 8 MPa or above. The results obtained in this work suggested that complexation with Folin reagent may be a rapid, sensitive and accurate method for determining rosmarinic acid in purified extracts from lemon balm leaves or from solid residues of supercritical extraction, but no direct relation was found between antioxidant effect and total phenol content of the extracts, suggesting that, in addition to the well known antioxidants, other compounds are also able to act as antioxidant. The extraction performed from supercritical solid residues was faster and easier than the extraction performed from lemon balm plant, and presents a high antioxidant activity. This can be
explained because the supercritical extraction had already extracted lipids and other high molecular weight compounds that make the extraction of polyphenols difficult. The extracts obtained in the first and second collector were analysed by rancimat method and all of them presented a protection factor lower or equal to one, perhaps due to the catalytic effect of chlorophyll, which has a pro-oxidant effect. It is assumed that we are at optimum extraction condition when the best protection factor curve of the supercritical solid residues (ASR) was obtained, and this is attributed for the lemon balm plant at conditions of 10 MPa, 308 K and 4 h. The results obtained in this work indicate that the supercritical extraction could be an effective way of concentrating the antioxidant in solid materials at low pressures.
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Acknowledgements This work was sponsored by PAMAF through project D068-AROMED.
[8]
[9]
References [1] T. Adzet, R. Ponz, E. Wolf, E. Schulte, Content and composition of M. officinalis oil in relation to leaf position and harvest time, Planta Med. 58 (1992) 562. [2] U. Hener, S. Faulhaber, P. Kreis, A. Mosandl, On the authenticity evaluation of balm oil (Melissa officinalis L.), Pharmazie 50 (1995) 60. [3] I. Nykanen, L. Nykanen, Flavour composition of lemon balm (Melissa officinalis L.) cultivated in Finland, Lebensm.-Wiss. Technol. 19 (1986) 482. [4] Y. Endo, R. Usuki, T. Kaneda, Antioxidant effects of chlorophyll and pheophytin on the autoxidation of oils in the dark. II. The mechanism of antioxidative action of chlorophyll, J. Am. Oil Chem. Soc. 62 (1985) 1387. [5] F. Shahidi, R.B. Pegg, Hexanal as an indicator of meat flavor deterioration, J. Food Lipids 1 (1994) 177. [6] U.N. Wanasundara, F. Shahidi, Antioxidant and pro-oxidant activity of green tea extracts in marine oils, Food Chem. 63 (1998) 335. [7] H.L. Madsen, B. Sorensen, L.H. Skibsted, G. Bertelsen, The antioxidant activity of summer savory (Satureja hortensis L.) and rosemary (Rosmarinus officinalis L.) in dress-
[10]
[11]
[12]
[13]
.
ing stored exposed to light or in darkness, Food Chem. 63 (1998) 173. T. Lo´ pez-Arnoldos, M. Lo´ pez-Serrano, A. Ros Barcelo´ , A.A. Caldero´ n, J.M. Zapata, Spectrophotometric determination of rosmarinic acid in plant cell cultures by complexation with Fe2 + ions, Fresenius J. Anal. Chem. 351 (1995) 311. M. Perrut, Supercritical fluid applications: industrial developments and economic issues, Proceedings of the fifth Conference on Supercritical Fluids and their Applications, AIDIC, 1999, p. 1. N. Sanders, Food legislation and the scope for increased use of near-critical fluid extraction operations in the food, flavouring and pharmaceutical industries, in: M.B. King, T.R. Bott (Eds.), Extraction of Natural Products using Near-Critical Solvents, Chapman & Hall, London, 1993, p. 34. M.M. Esquivel, M.A. Ribeiro, M.G. Bernardo-Gil, Supercritical extraction of savory oil: study of antioxidant activity and extract characterization, J. Supercritical Fluids 14 (1999) 129. R.M. Duran, R.B. Padilla, A.M. Martin, J.A.F. Ros de Ursinos, J.A. Mendonza, Biodegradacio´ n de los compuestos feno´ licos presentes en el alpechı´n, Grasas Aceites 42 (1991) 271. S.B. Hawthorne, M.L. Riekkola, K. Serenius, Y. Holm, R. Hiltunen, K. Hartonen, Comparison of hydrodistillation and supercritical fluid extraction for the determination of essential oils in aromatic plants, J. Chromatogr. 634 (1993) 297.
Melissa officinalis, L.: study of antioxidant activity in supercritical residues M.A. Ribeiro *, M.G. Bernardo-Gil, M.M. Esquı´vel Centro de Engenharia Biolo´gica e Quimica, Departamento de Engenharia Quı´mica, Instituto Superior Te´cnico, A6. Ro6isco Pais, 1049 -001 Lisboa, Portugal Received 4 May 2000; received in revised form 1 March 2001; accepted 13 March 2001
Abstract The supercritical CO2 extraction of lemon balm (Melissa officinalis, L.) at pressures from 10 to 18 MPa and at temperatures of 308–313 K was studied. The antioxidant activity of lemon balm extracts, obtained from solid residues of supercritical extraction and from raw lemon balm leaves, was performed using the Rancimat method. The best protection factor curve was obtained when extracts from the solid residues of supercritical extraction at 10 MPa, 308 K and 4 h of extraction time were used. A spectrophotometric method was used for the determination of the polyphenol compounds in the extraction residues. The highest value of phenol compounds was obtained for the extracts of solid residues of supercritical extraction at 10 MPa, 323 K and 30 min. © 2001 Elsevier Science B.V. All rights reserved. Keywords: Antioxidant; Lemon balm; Polyphenol; Rancimat; Supercritical extraction
1. Introduction Lemon balm (Melissa officinalis, L.) is known as an officinal herb of a long tradition and a large variety of uses. Many of the therapeutic effects of this herb are attributed to its leaf essential oil, which is rich in aldehydes and terpenic alcohols. The main constituents are citral (geranial and neral), citronellal, linalool, geraniol, b-caryophyllene and b-caryophyllene oxide, comprising about 96% of the oil ingredients [1]. Therefore, it is not * Corresponding author. Tel.: + 351-21-8417312; Fax: + 351-21-8499246. E-mail address: [email protected] (M.A. Ribeiro).
surprising that lemon balm oil has a special reputation among essential oils. As a result of the low content of essential oil (balm leaves normally contain 0.1% approximately), balm oil has a very high price level [2]. The differences encountered between the oils obtained from different origin countries from dried whole plants or dried leaves are in the yield, rather than in the qualitative composition [3]. Generally, the antioxidant effect has been explained by the two following mechanisms. One mechanism is ascribed to scavenging free radicals such as peroxy and alkyl radicals to break the chain reaction. Another one is ascribed to the decomposition of hydroperoxides [4].
0896-8446/01/$ - see front matter © 2001 Elsevier Science B.V. All rights reserved. PII: S 0 8 9 6 - 8 4 4 6 ( 0 1 ) 0 0 0 7 8 - X
52
M.A. Ribeiro et al. / J. of Supercritical Fluids 21 (2001) 51–60
The oxidation of unsaturated lipids has been extensively studied since it relates to deterioration of muscle foods, production of both desirable and undesirable breakdown products, and numerous reactions associated with other food constituents [5]. Synthetic phenolic compounds, such as butylated hydroxyanisole (BHA), butylated hydroxytoluene (BHT) and tert-butylhydroquinone (TBHQ), have been widely used as antioxidants in food lipids. However, possible toxicity and/or mutagenecity of these antioxidants have been a subject of study for many years. There are several recent reports on the effect of BHA on conversion of ingested material into toxic substances or carcinogens due to increase secretion of microsomal enzymes of liver and extra hepatic organs, such as the lungs and gastrointestinal tract mucosa. Therefore, at the present time, the Food and Drug Administration in the US is examining possible removal of BHA from the Generally Recognised As Safe list [6]. Furthermore, having been reported that BHT is carcinogenic in rats, this antioxidant is also in the process of being carefully scrutinised. In addition, TBHQ has not been approved for food use in Europe, Japan and Canada. Thus, natural antioxidants have gained popularity in recent years [6]. Chlorophyll is an important photosensitizer in plant-derived foods, although the presence of carotenoids in the chloroplast normally yields protection by quenching of singlet oxygen. This protection may, however, be impaired by drying and extraction of herbs. Exposures to light change this antioxidative effect to a pro-oxidative effect [7]. For storage in light, a relatively poor antioxidative effect has been seen for plants with higher quantities of chlorophyll. Naturally occurring antioxidative components in foods include flavonoids, phenolic acids, lignan percursors, terpenes, mixed tocopherols, phospholipids, polyfunctional organic acids and also plant extracts. The compounds in lemon balm (M. officinalis) that showed antioxidant activity, caffeic acid and rosmarinic acid, were more active than a-tocopherol, having an activity comparable with the BHA.
Rosmarinic acid (a-o-caffeoyl-3,4-dihydroxyphenallatic acid) is one of the most abundant caffeic acid esters occurring in plants. It is mainly found in species of the Boraginaceae and Lamiaceae families, and is noted for its potent antioxidant properties [8]. Current research on rosmarinic acid centres on its physiological and pharmacological activities. Oxidised rosmarinic acid has displayed antithyrotropic activity in testes with human thyroid membrane preparations, and the pure compound has been shown to effectively suppress the complement-dependent components of endo toxin shock in rabbits. Rosmarinic acid is also known to react rapidly with viral coat proteins and so inactivate the virus. There seems to be sufficient promise as regards its useful properties to have led at least one pharmaceutical firm to undertake a serious examination of rosmarinic acid as a potential pharmaceutical plant product [8]. The majority of extractions carried out for the food, flavouring and perfumery industries will be on solid vegetable starting materials. Plant materials usually have a low bulk density (typically 500 kg m − 3) and a fairly low extractable content, which obliges the use of large volume extractors. Supercritical fluid technology is often considered expensive due to very high investment costs in comparison with classical low-pressure equipment. Most companies believe that it leads to high-quality products, but it is in fact restricted to high-added value products. However, this is far from true when very large volumes of materials are treated [9]. Carbon dioxide is a very non-polar solvent with characteristics similar to pentane or hexane. Commercial CO2 is obtained as a by-product of fermentation processes, so its use as an extraction solvent does not increase the amount already present in the atmosphere. Therefore, there is no overall detrimental effect on the earth’s ozone layer from this use of CO2 [10]. Although extensive R&D investigations have been carried out world wide for more than 25 years, it is disappointing that supercritical fluid applications have been still limited to few areas; and it does not appear that development will rocket in the near future. However, this should
M.A. Ribeiro et al. / J. of Supercritical Fluids 21 (2001) 51–60
not lead one to give up the numerous opportunities arising now, from food ingredients and nutraceuticals to pharmaceuticals, from biological applications and pollution abatement to new material manufacture [9]. The aim of this work was to obtain extracts with antioxidant power. Two solid materials were studied: 1. lemon balm leaves; and 2. the solid residues obtained after the supercritical extraction of the oil. Parameters such as pressure, temperature and extraction time were studied. The pressure of the first collector was also analysed. The influence of theses parameters on the antioxidant activity of the extracts (ASR) was performed using the Rancimat method. Determination of the polyphenols compounds in the extraction residue was carried out using a spectrophotometric method.
2. Materials and methods Lemon balm was collected in Portugal and was used as received (air-dried and coarsely cut). The CO2 used in this work was 99.5% pure (w/w), and was supplied by Ar Liquido (Portugal). The standard Rosmarinic acid was purchased from Extrasynthese (Genay, France). All the other chemicals used in this work were obtained from various commercial suppliers and were of the highest purity available.
2.1. Supercritical extraction The main interest of the supercritical extraction experiments was on the solid residue of the extraction, to be used as raw material for antioxidant extraction. Extraction measurements were carried out in a semi-batch flow extraction apparatus. The supercritical extraction apparatus has been described elsewhere [11]. It mainly consisted of a 500 ml extractor (L/D =5.8), two 200 ml separation vessels type cyclones SFE500 (Separex, Champigneulles, France), operated in series and operating at different pressures and temperatures, called in this
53
work the first and second collectors. A glass coil (immersed in a dry ice/acetone bath) was placed at the exit of the apparatus at about 203 K and at ambient pressure. The volume of carbon dioxide delivered was measured by a Dry Test Metter (American Meter Company, Philadelphia, USA).
2.2. Antioxidant extraction 2.2.1. Preparation of lemon balm extracts The ground lemon balm leaves and ground supercritical extraction solid residues (Moulinex coffee grinder) were boiled with distilled water for about 1.5 h, using an agitation plate and a magnetic stirrer. The homogenate was then filtered through a coffee filter paper; the collected filtrate was acidulated with a solution of 25% HCl to pH 2.5 to precipitate the waxes. After filtration with a Whatman number 1 filter paper, the aqueous phase was mixed with diisopropilether (10:3) and allowed to separate. The upper aqueous layer was extracted twice. The organic phases were combined and dryness with MgSO4, anhydrous filtered and then evaporated using a rotary evaporator (Heidolph VV2000). Schemes of the main operations for the preparation of lemon balm antioxidant from supercritical residues (ASR) and from raw lemon balm leaves (ARM) are shown in Figs. 1 and 2, respectively. 2.3. Polyphenol determination A quantitative determination of total phenols by a spectrophotometric method has been carried out using a spectrophotometer (Unicam Helios a). The antioxidant extracted with diisopropilether was dissolved in a mixture of methanol:water (6:4) to obtain a solution of 100 p.p.m. in antioxidant extract. On a volumetric flask of 25 ml has been put 17.5 ml distilled water, 1 ml solution methanol:water (6:4) and 1.25 ml Folin reagent (mixture of phosphomolibdic and phosphowolframic acid). The solution was agitated and rested for 3 min, after which was added 2.5 ml Na2CO3
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solution (20%), and the volume of the flask completed with distilled water.
The absorbance at 750 nm was measured after 1 h in a dark camera. The spectrophotometric calibration curve with different concentrations of standard rosmarinic acid has been carried out. The white and the standard solutions were prepared in the same way but without added polyphenol solution (in the first case) and with an added 1 ml rosmarinic acid standard solution with a concentration of 50 p.p.m. (in the second case) [12]. The results were expressed in rosmarinic acid because it is one of the major compounds present in lemon balm, and all the extractions and purification were carried out to obtain a purified rosmarinic acid.
2.4. Rancimat method
Fig. 1. Scheme for the preparation of antioxidant extracts from supercritical residues of lemon balm (ASR).
The rancimat method (Metrohm Rancimat 679) was used for the determination of the antioxidant activity. Samples of extracts dissolved in sunflower oil at a concentration range from 200 to 4000 p.p.m. were heated at 393 K. A continuous air stream (20 l h − 1) at ambient condition was passed through the heated samples and the volatile compounds were absorbed in a conductivity cell. The conductivity was monitored continuously until a sudden rise signified the end of the induction period [11].
3. Results and discussion
Fig. 2. Scheme for the preparation of antioxidant extracts from raw material (ARM).
Antioxidant extract yield from raw material, lemon balm leaves (ARM) was 0.4 g extract/100 g plant material. When the ground supercritical solid residues (ASR) were used, yields of 0.6 g extract/100 g supercritical solid residue were obtained, independently of the extraction pressure. The interest of this work was not the study of essential oil extraction or the study of its fractionation, although it was observed that the pressure in the first collector was of primordial importance for the total mass of extract recovered. As can be seen in Fig. 3, for the same pressure and temperature of the extraction, respectively, of 12 MPa and 313 K, and a superficial velocity of carbon dioxide of 0.02 cm s − 1 but different pres-
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Fig. 3. Percentage of solute recovered for a extraction condition of 12 MPa, 313 K, a superficial velocity of carbon dioxide of 0.02 cms − 1 and different pressures of the first collector: , 8–9 MPa; , 6 – 7MPa.
sure of the first collector, the amount of extract collected was quite different. High recoveries (30%) were found when the pressure of the first collector was 8–9 MPa, but only 8% (w/w) were obtained when the pressure of the first collector varies between 6 and 7 MPa. The percentage of solute collected was calculated by dividing the total mass of solute collected by the weight of material charged in the extractor. In the first collector, the cuticular waxes were selectively precipitated, which is normal because the alkanes are extracted more rapidly with pure CO2 than the essential oil components, which might be expected since plant waxes are found on the tissue surface. However, for pressures under 8 MPa, the high molecular weight compounds could not be recovered because of the poor collection efficiencies, which result in low recoveries and could mistakenly be blamed on poor extraction efficiencies [13].
The spectophotometric polyphenol determination was expressed as the percentage of rosmarinic acid in antioxidant extracts of M. officinalis, because it is one of the major compounds present in lemon balm, and the experiments were carried out to perform a purification of rosmarinic acid. This concentration depends on the extraction conditions used, varying between 10 and 80%, as presented in Fig. 4. The highest concentration of phenol compounds was observed for the solid residues obtained at 10 MPa, 323 K and 0.5 h of extraction. For this extraction condition, the oily extract was green, having a great quantity of chlorophyll, which was confirmed spectrophotometrically at 600 nm. If great quantities of chlorophyll were extracted, the remaining amount of unwanted compounds that can minimize or hide the polyphenol determination in solid residues was
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lower and, subsequently, the phenol percentage was higher. The protection factor (PF) was calculated by dividing the induction time of the sample by the induction time of sunflower oil. When materials do not have antioxidant activity, the induction time of their dispersion is equal to the induction time of the control sample (sunflower oil), and PF is equal to 1. A PF greater than 1 indicates antioxidant activity, while a PF less than 1 indicates pro-oxidant activity. In Figs. 5–7, the protection factors of extracts were plotted as a function of extract concentration (ASR) in sunflower oil at different pressures but at constant extraction times of 0.5, 4, and 20 h, respectively. For a short extraction time of 0.5
h, the antioxidant activity of the ASR extracts was not greatly affected by the pressure and temperature, but for higher extraction times the behaviour of the sample was different. These two different trends seem to be justified taking into account that, for the shorter extraction time 0.5 h, the only extractable compounds were volatile compounds and cuticular waxes, located on the surface of the vegetable matter, and the extraction conditions did not seem to interfere significantly with the extraction of components that influence the antioxidant activity of the supercritical solid residues (ASR). But this was not true for the high extraction time of 4 h, where the balance between solvent power and selectivity must be pondered. High densities induce high
Fig. 4. Percentage of phenol on extracts of supercritical solid residues (ASR), and for the extract of M. officinalis (ARM).
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Fig. 5. Protection factor versus concentration of antioxidant extracts (ASR) for an extraction time of 0.5 h, a superficial velocity of carbon dioxide of 0.02 cm s − 1, and different conditions of pressure and temperature: , 18 MPa, 323 K; *, 10 MPa, 308 K; , 10 MPa, 323 K.
solvent power and low selectivity. The balance between these two important factors, solvent power and selectivity of the supercritical CO2 is perhaps the most important role to obtain a supercritical solid residue with higher antioxidant activity. Natural materials contain various extractable materials. Among these, the waxes greatly hamper the preparation of antioxidant extracts because the filtration of the water solution (ground leaves boiled with distillate water) was quite difficult. Without these types of compounds, the filtration time will be much improved. It was possible to spare heaps of time. For the long extraction time of 20 h, as can be seen in Fig. 7, the antioxidant activity was lower than for shorter extraction time (Figs. 5 and 6) at the same concentration of solution. For long extraction times, compounds with great chains like flavonoids, flavonoics, triterpe-
nes, and organic acids began to be extracted. These classes of compounds typical of oleoresins show a very stable antioxidant activity when in synergy with phenol compounds. If these compounds are extracted, its concentration in the residue is lower and, subsequently, the stability of the antioxidant activity is affected. This is the reason why, at the same concentration, extracts obtained from long extraction times show lower antioxidant activity. Besides, if the extraction pressure increase for the same conditions of temperature and velocity of carbon dioxide, the solvent power increase and, consequently, the antioxidant activity of the residue decrease, because as has already been said the compounds with great chains began to be extracted. On a closer view of the Fig. 8, the antioxidant activity of extracts obtained from supercritical extraction residues of lemon balm increased; all of
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them with increasing concentrations of the extracts. But, as has been previously discussed, the behaviour of the extracts changed at an aleatory form,
independent of the extraction conditions used. The best curve was obtained for the condition of 10 MPa, 308 K and 4 h of extraction.
Fig. 6. Protection factor versus concentration of antioxidant extract (ASR) for an extraction time of 4 h, a superficial velocity of carbon dioxide of 0.02 cm s − 1, and different conditions of pressure and temperature: *, 10 MPa, 308 K; , 10 MPa, 323 K; , 18 MPa, 323 K.
Fig. 7. Protection factor versus concentration of antioxidant extract (ASR) in sunflower oil for long extraction time, 20 h, and temperature of 313 K and a superficial velocity of CO2 of 0.02 cm s − 1: ", 12 MPa; ×, 18 MPa.
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Fig. 8. Protection factor versus concentration of antioxidant extract in sunflower oil: , extracted lemon balm (ARM); —, BHT; *, 10 MPa, 308 K, 4 h; , 18 MPa, 323 K, 0.5 h; , 12 MPa, 308 K, 2 h; ", 12 MPa, 313 K, 20 h; ×, 18 MPa, 313 K, 20 h;
, 10 MPa, 323 K, 0.5 h; , Tocobiol; , 10 MPa, 308 K, 0.5 h; , BHA; +, 18 MPa, 323 K, 4 h.
4. Conclusions The best pressures for the first collector, if we were not interested in essential oil and subsequently its fractionation, but only in the residue, are 8 MPa or above. The results obtained in this work suggested that complexation with Folin reagent may be a rapid, sensitive and accurate method for determining rosmarinic acid in purified extracts from lemon balm leaves or from solid residues of supercritical extraction, but no direct relation was found between antioxidant effect and total phenol content of the extracts, suggesting that, in addition to the well known antioxidants, other compounds are also able to act as antioxidant. The extraction performed from supercritical solid residues was faster and easier than the extraction performed from lemon balm plant, and presents a high antioxidant activity. This can be
explained because the supercritical extraction had already extracted lipids and other high molecular weight compounds that make the extraction of polyphenols difficult. The extracts obtained in the first and second collector were analysed by rancimat method and all of them presented a protection factor lower or equal to one, perhaps due to the catalytic effect of chlorophyll, which has a pro-oxidant effect. It is assumed that we are at optimum extraction condition when the best protection factor curve of the supercritical solid residues (ASR) was obtained, and this is attributed for the lemon balm plant at conditions of 10 MPa, 308 K and 4 h. The results obtained in this work indicate that the supercritical extraction could be an effective way of concentrating the antioxidant in solid materials at low pressures.
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Acknowledgements This work was sponsored by PAMAF through project D068-AROMED.
[8]
[9]
References [1] T. Adzet, R. Ponz, E. Wolf, E. Schulte, Content and composition of M. officinalis oil in relation to leaf position and harvest time, Planta Med. 58 (1992) 562. [2] U. Hener, S. Faulhaber, P. Kreis, A. Mosandl, On the authenticity evaluation of balm oil (Melissa officinalis L.), Pharmazie 50 (1995) 60. [3] I. Nykanen, L. Nykanen, Flavour composition of lemon balm (Melissa officinalis L.) cultivated in Finland, Lebensm.-Wiss. Technol. 19 (1986) 482. [4] Y. Endo, R. Usuki, T. Kaneda, Antioxidant effects of chlorophyll and pheophytin on the autoxidation of oils in the dark. II. The mechanism of antioxidative action of chlorophyll, J. Am. Oil Chem. Soc. 62 (1985) 1387. [5] F. Shahidi, R.B. Pegg, Hexanal as an indicator of meat flavor deterioration, J. Food Lipids 1 (1994) 177. [6] U.N. Wanasundara, F. Shahidi, Antioxidant and pro-oxidant activity of green tea extracts in marine oils, Food Chem. 63 (1998) 335. [7] H.L. Madsen, B. Sorensen, L.H. Skibsted, G. Bertelsen, The antioxidant activity of summer savory (Satureja hortensis L.) and rosemary (Rosmarinus officinalis L.) in dress-
[10]
[11]
[12]
[13]
.
ing stored exposed to light or in darkness, Food Chem. 63 (1998) 173. T. Lo´ pez-Arnoldos, M. Lo´ pez-Serrano, A. Ros Barcelo´ , A.A. Caldero´ n, J.M. Zapata, Spectrophotometric determination of rosmarinic acid in plant cell cultures by complexation with Fe2 + ions, Fresenius J. Anal. Chem. 351 (1995) 311. M. Perrut, Supercritical fluid applications: industrial developments and economic issues, Proceedings of the fifth Conference on Supercritical Fluids and their Applications, AIDIC, 1999, p. 1. N. Sanders, Food legislation and the scope for increased use of near-critical fluid extraction operations in the food, flavouring and pharmaceutical industries, in: M.B. King, T.R. Bott (Eds.), Extraction of Natural Products using Near-Critical Solvents, Chapman & Hall, London, 1993, p. 34. M.M. Esquivel, M.A. Ribeiro, M.G. Bernardo-Gil, Supercritical extraction of savory oil: study of antioxidant activity and extract characterization, J. Supercritical Fluids 14 (1999) 129. R.M. Duran, R.B. Padilla, A.M. Martin, J.A.F. Ros de Ursinos, J.A. Mendonza, Biodegradacio´ n de los compuestos feno´ licos presentes en el alpechı´n, Grasas Aceites 42 (1991) 271. S.B. Hawthorne, M.L. Riekkola, K. Serenius, Y. Holm, R. Hiltunen, K. Hartonen, Comparison of hydrodistillation and supercritical fluid extraction for the determination of essential oils in aromatic plants, J. Chromatogr. 634 (1993) 297.