melissa officinalis, l.: study of antioxidant activity in supercritical residues

Journal of Supercritical Fluids 21 (2001) 51–60

Melissa officinalis, L.: study of antioxidant activity insupercritical residues

M.A. Ribeiro *, M.G. Bernardo-Gil, M.M. EsquıvelCentro de Engenharia Biologica e Quimica, Departamento de Engenharia Quımica, Instituto Superior Tecnico, A�. Ro�isco 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 attemperatures of 308–313 K was studied. The antioxidant activity of lemon balm extracts, obtained from solidresidues of supercritical extraction and from raw lemon balm leaves, was performed using the Rancimat method. Thebest 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 thepolyphenol compounds in the extraction residues. The highest value of phenol compounds was obtained for theextracts of solid residues of supercritical extraction at 10 MPa, 323 K and 30 min. © 2001 Elsevier Science B.V. Allrights reserved.

Keywords: Antioxidant; Lemon balm; Polyphenol; Rancimat; Supercritical extraction

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1. Introduction

Lemon balm (Melissa officinalis, L.) is knownas an officinal herb of a long tradition and a largevariety of uses. Many of the therapeutic effects ofthis herb are attributed to its leaf essential oil,which is rich in aldehydes and terpenic alcohols.The main constituents are citral (geranial andneral), citronellal, linalool, geraniol, �-caryophyl-lene and �-caryophyllene oxide, comprising about96% of the oil ingredients [1]. Therefore, it is not

surprising that lemon balm oil has a special repu-tation among essential oils. As a result of the lowcontent of essential oil (balm leaves normallycontain 0.1% approximately), balm oil has a veryhigh price level [2]. The differences encounteredbetween the oils obtained from different origincountries from dried whole plants or dried leavesare in the yield, rather than in the qualitativecomposition [3].

Generally, the antioxidant effect has been ex-plained by the two following mechanisms. Onemechanism is ascribed to scavenging free radicalssuch as peroxy and alkyl radicals to break thechain reaction. Another one is ascribed to thedecomposition of hydroperoxides [4].

* Corresponding author. Tel.: +351-21-8417312; Fax: +351-21-8499246.

E-mail address: [email protected] (M.A. Ribeiro).

0896-8446/01/$ - see front matter © 2001 Elsevier Science B.V. All rights reserved.

PII: S 0896 -8446 (01 )00078 -X

M.A. Ribeiro et al. / J. of Supercritical Fluids 21 (2001) 51–6052

The oxidation of unsaturated lipids has beenextensively studied since it relates to deteriorationof muscle foods, production of both desirable andundesirable breakdown products, and numerousreactions associated with other food constituents[5].

Synthetic phenolic compounds, such as buty-lated hydroxyanisole (BHA), butylated hydroxy-toluene (BHT) and tert-butylhydroquinone(TBHQ), have been widely used as antioxidants infood lipids. However, possible toxicity and/or mu-tagenecity of these antioxidants have been a sub-ject of study for many years.

There are several recent reports on the effect ofBHA on conversion of ingested material intotoxic substances or carcinogens due to increasesecretion of microsomal enzymes of liver andextra hepatic organs, such as the lungs and gas-trointestinal tract mucosa. Therefore, at thepresent time, the Food and Drug Administrationin the US is examining possible removal of BHAfrom the Generally Recognised As Safe list [6].

Furthermore, having been reported that BHT iscarcinogenic in rats, this antioxidant is also in theprocess of being carefully scrutinised. In addition,TBHQ has not been approved for food use inEurope, Japan and Canada. Thus, natural antiox-idants have gained popularity in recent years [6].

Chlorophyll is an important photosensitizer inplant-derived foods, although the presence ofcarotenoids in the chloroplast normally yieldsprotection by quenching of singlet oxygen. Thisprotection may, however, be impaired by dryingand extraction of herbs. Exposures to light changethis antioxidative effect to a pro-oxidative effect[7].

For storage in light, a relatively poor antioxida-tive effect has been seen for plants with higherquantities of chlorophyll.

Naturally occurring antioxidative componentsin foods include flavonoids, phenolic acids, lignanpercursors, terpenes, mixed tocopherols, phospho-lipids, polyfunctional organic acids and also plantextracts.

The compounds in lemon balm (M. officinalis)that showed antioxidant activity, caffeic acid androsmarinic acid, were more active than �-toco-pherol, having an activity comparable with theBHA.

Rosmarinic acid (�-o-caffeoyl-3,4-dihydrox-yphenallatic acid) is one of the most abundantcaffeic acid esters occurring in plants. It is mainlyfound in species of the Boraginaceae and Lami-aceae families, and is noted for its potent antioxi-dant properties [8].

Current research on rosmarinic acid centres onits physiological and pharmacological activities.Oxidised rosmarinic acid has displayed antithyro-tropic activity in testes with human thyroid mem-brane preparations, and the pure compound hasbeen shown to effectively suppress the comple-ment-dependent components of endo toxin shockin rabbits. Rosmarinic acid is also known to reactrapidly with viral coat proteins and so inactivatethe virus. There seems to be sufficient promise asregards its useful properties to have led at leastone pharmaceutical firm to undertake a seriousexamination of rosmarinic acid as a potentialpharmaceutical plant product [8].

The majority of extractions carried out for thefood, flavouring and perfumery industries will beon solid vegetable starting materials. Plant materi-als usually have a low bulk density (typically 500kg m−3) and a fairly low extractable content,which obliges the use of large volume extractors.

Supercritical fluid technology is often consid-ered expensive due to very high investment costsin comparison with classical low-pressure equip-ment. Most companies believe that it leads tohigh-quality products, but it is in fact restricted tohigh-added value products. However, this is farfrom true when very large volumes of materialsare treated [9].

Carbon dioxide is a very non-polar solvent withcharacteristics similar to pentane or hexane. Com-mercial CO2 is obtained as a by-product of fer-mentation processes, so its use as an extractionsolvent does not increase the amount alreadypresent in the atmosphere. Therefore, there is nooverall detrimental effect on the earth’s ozonelayer from this use of CO2 [10].

Although extensive R&D investigations havebeen carried out world wide for more than 25years, it is disappointing that supercritical fluidapplications have been still limited to few areas;and it does not appear that development willrocket in the near future. However, this should

M.A. Ribeiro et al. / J. of Supercritical Fluids 21 (2001) 51–60 53

not lead one to give up the numerous opportuni-ties arising now, from food ingredients and nu-traceuticals to pharmaceuticals, from biologicalapplications and pollution abatement to new ma-terial manufacture [9].

The aim of this work was to obtain extractswith antioxidant power. Two solid materials werestudied:1. lemon balm leaves; and2. the solid residues obtained after the supercriti-

cal extraction of the oil.Parameters such as pressure, temperature and

extraction time were studied. The pressure of thefirst collector was also analysed. The influence oftheses parameters on the antioxidant activity ofthe extracts (ASR) was performed using theRancimat method.

Determination of the polyphenols compoundsin the extraction residue was carried out using aspectrophotometric method.

2. Materials and methods

Lemon balm was collected in Portugal and wasused as received (air-dried and coarsely cut). TheCO2 used in this work was 99.5% pure (w/w), andwas supplied by Ar Liquido (Portugal). The stan-dard Rosmarinic acid was purchased from Ex-trasynthese (Genay, France). All the otherchemicals used in this work were obtained fromvarious commercial suppliers and were of thehighest purity available.

2.1. Supercritical extraction

The main interest of the supercritical extractionexperiments was on the solid residue of the extrac-tion, to be used as raw material for antioxidantextraction.

Extraction measurements were carried out in asemi-batch flow extraction apparatus. The super-critical extraction apparatus has been describedelsewhere [11]. It mainly consisted of a 500 mlextractor (L/D=5.8), two 200 ml separation ves-sels type cyclones SFE500 (Separex, Champigneu-lles, France), operated in series and operating atdifferent pressures and temperatures, called in this

work the first and second collectors. A glass coil(immersed in a dry ice/acetone bath) was placedat the exit of the apparatus at about 203 K and atambient pressure.

The volume of carbon dioxide delivered wasmeasured by a Dry Test Metter (American MeterCompany, Philadelphia, USA).

2.2. Antioxidant extraction

2.2.1. Preparation of lemon balm extractsThe ground lemon balm leaves and ground

supercritical extraction solid residues (Moulinexcoffee grinder) were boiled with distilled water forabout 1.5 h, using an agitation plate and a mag-netic stirrer. The homogenate was then filteredthrough a coffee filter paper; the collected filtratewas acidulated with a solution of 25% HCl to pH2.5 to precipitate the waxes. After filtration with aWhatman number 1 filter paper, the aqueousphase was mixed with diisopropilether (10:3) andallowed to separate. The upper aqueous layer wasextracted twice. The organic phases were com-bined and dryness with MgSO4, anhydrousfiltered and then evaporated using a rotary evapo-rator (Heidolph VV2000).

Schemes of the main operations for the prepa-ration of lemon balm antioxidant from supercriti-cal residues (ASR) and from raw lemon balmleaves (ARM) are shown in Figs. 1 and 2,respectively.

2.3. Polyphenol determination

A quantitative determination of total phenolsby a spectrophotometric method has been carriedout using a spectrophotometer (Unicam He�ios�).

The antioxidant extracted with diisopropiletherwas dissolved in a mixture of methanol:water (6:4)to obtain a solution of 100 p.p.m. in antioxidantextract.

On a volumetric flask of 25 ml has been put17.5 ml distilled water, 1 ml solutionmethanol:water (6:4) and 1.25 ml Folin reagent(mixture of phosphomolibdic and phosphowol-framic acid). The solution was agitated and restedfor 3 min, after which was added 2.5 ml Na2CO3


solution (20%), and the volume of the flask com-pleted with distilled water.

The absorbance at 750 nm was measured after1 h in a dark camera. The spectrophotometriccalibration curve with different concentrations ofstandard rosmarinic acid has been carried out.The white and the standard solutions were pre-pared in the same way but without addedpolyphenol solution (in the first case) and with anadded 1 ml rosmarinic acid standard solution witha concentration of 50 p.p.m. (in the second case)[12].

The results were expressed in rosmarinic acidbecause it is one of the major compounds presentin lemon balm, and all the extractions and purifi-cation were carried out to obtain a purified ros-marinic acid.

2.4. Rancimat method

The rancimat method (Metrohm Rancimat 679)was used for the determination of the antioxidantactivity. Samples of extracts dissolved insunflower oil at a concentration range from 200 to4000 p.p.m. were heated at 393 K. A continuousair stream (20 l h−1) at ambient condition waspassed through the heated samples and thevolatile compounds were absorbed in a conductiv-ity cell. The conductivity was monitored continu-ously until a sudden rise signified the end of theinduction period [11].

3. Results and discussion

Antioxidant extract yield from raw material,lemon balm leaves (ARM) was 0.4 g extract/100 gplant material. When the ground supercriticalsolid residues (ASR) were used, yields of 0.6 gextract/100 g supercritical solid residue were ob-tained, independently of the extraction pressure.

The interest of this work was not the study ofessential oil extraction or the study of its fraction-ation, although it was observed that the pressurein the first collector was of primordial importancefor the total mass of extract recovered.

As can be seen in Fig. 3, for the same pressureand temperature of the extraction, respectively, of12 MPa and 313 K, and a superficial velocity ofcarbon dioxide of 0.02 cm s−1 but different pres-

Fig. 1. Scheme for the preparation of antioxidant extractsfrom supercritical residues of lemon balm (ASR).

Fig. 2. Scheme for the preparation of antioxidant extractsfrom raw material (ARM).


Fig. 3. Percentage of solute recovered for a extraction condition of 12 MPa, 313 K, a superficial velocity of carbon dioxide of 0.02cms−1 and different pressures of the first collector: �, 8–9 MPa; �, 6–7MPa.

sure of the first collector, the amount of extractcollected was quite different. High recoveries(30%) were found when the pressure of the firstcollector was 8–9 MPa, but only 8% (w/w) wereobtained when the pressure of the first collectorvaries between 6 and 7 MPa. The percentage ofsolute collected was calculated by dividing thetotal mass of solute collected by the weight ofmaterial charged in the extractor.

In the first collector, the cuticular waxes wereselectively precipitated, which is normal becausethe alkanes are extracted more rapidly with pureCO2 than the essential oil components, whichmight be expected since plant waxes are found onthe tissue surface. However, for pressures under 8MPa, the high molecular weight compoundscould not be recovered because of the poor collec-tion efficiencies, which result in low recoveries andcould mistakenly be blamed on poor extractionefficiencies [13].

The spectophotometric polyphenol determina-tion was expressed as the percentage of rosmarinicacid in antioxidant extracts of M. officinalis, be-cause it is one of the major compounds present inlemon balm, and the experiments were carried outto perform a purification of rosmarinic acid. Thisconcentration depends on the extraction condi-tions used, varying between 10 and 80%, as pre-sented in Fig. 4.

The highest concentration of phenol com-pounds was observed for the solid residues ob-tained at 10 MPa, 323 K and 0.5 h of extraction.For this extraction condition, the oily extract wasgreen, having a great quantity of chlorophyll,which was confirmed spectrophotometrically at600 nm. If great quantities of chlorophyll wereextracted, the remaining amount of unwantedcompounds that can minimize or hide thepolyphenol determination in solid residues was


lower and, subsequently, the phenol percentagewas higher.

The protection factor (PF) was calculated bydividing the induction time of the sample by theinduction time of sunflower oil. When materialsdo not have antioxidant activity, the inductiontime of their dispersion is equal to the inductiontime of the control sample (sunflower oil), and PFis equal to 1. A PF greater than 1 indicatesantioxidant activity, while a PF less than 1 indi-cates pro-oxidant activity.

In Figs. 5–7, the protection factors of extractswere plotted as a function of extract concentra-tion (ASR) in sunflower oil at different pressuresbut at constant extraction times of 0.5, 4, and 20h, respectively. For a short extraction time of 0.5

h, the antioxidant activity of the ASR extractswas not greatly affected by the pressure and tem-perature, but for higher extraction times the be-haviour of the sample was different.

These two different trends seem to be justifiedtaking into account that, for the shorter extrac-tion time 0.5 h, the only extractable compoundswere volatile compounds and cuticular waxes, lo-cated on the surface of the vegetable matter, andthe extraction conditions did not seem to interferesignificantly with the extraction of componentsthat influence the antioxidant activity of the su-percritical solid residues (ASR). But this was nottrue for the high extraction time of 4 h, where thebalance between solvent power and selectivitymust 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).


Fig. 5. Protection factor versus concentration of antioxidant extracts (ASR) for an extraction time of 0.5 h, a superficial velocity ofcarbon 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 balancebetween these two important factors, solventpower and selectivity of the supercritical CO2 isperhaps the most important role to obtain a su-percritical solid residue with higher antioxidantactivity.

Natural materials contain various extractablematerials. Among these, the waxes greatly hamperthe preparation of antioxidant extracts becausethe filtration of the water solution (ground leavesboiled with distillate water) was quite difficult.Without these types of compounds, the filtrationtime will be much improved. It was possible tospare heaps of time.

For the long extraction time of 20 h, as can beseen in Fig. 7, the antioxidant activity was lowerthan for shorter extraction time (Figs. 5 and 6) atthe same concentration of solution.

For long extraction times, compounds withgreat chains like flavonoids, flavonoics, triterpe-

nes, and organic acids began to be extracted.These classes of compounds typical of oleoresinsshow a very stable antioxidant activity when insynergy with phenol compounds. If these com-pounds are extracted, its concentration in theresidue is lower and, subsequently, the stability ofthe antioxidant activity is affected. This is thereason why, at the same concentration, extractsobtained from long extraction times show lowerantioxidant activity. Besides, if the extractionpressure increase for the same conditions of tem-perature and velocity of carbon dioxide, the sol-vent power increase and, consequently, theantioxidant activity of the residue decrease, be-cause as has already been said the compoundswith great chains began to be extracted.

On a closer view of the Fig. 8, the antioxidantactivity of extracts obtained from supercriticalextraction residues of lemon balm increased; all of


them with increasing concentrations of the extracts.But, as has been previously discussed, the be-haviour 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 ofcarbon 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, andtemperature of 313 K and a superficial velocity of CO2 of 0.02 cm s−1: �, 12 MPa; × , 18 MPa.


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 wewere not interested in essential oil and subse-quently its fractionation, but only in the residue,are 8 MPa or above.

The results obtained in this work suggestedthat complexation with Folin reagent may be arapid, sensitive and accurate method for deter-mining rosmarinic acid in purified extracts fromlemon balm leaves or from solid residues of su-percritical extraction, but no direct relation wasfound between antioxidant effect and total phe-nol content of the extracts, suggesting that, inaddition to the well known antioxidants, othercompounds are also able to act as antioxidant.

The extraction performed from supercriticalsolid residues was faster and easier than the ex-traction performed from lemon balm plant, andpresents a high antioxidant activity. This can be

explained because the supercritical extractionhad already extracted lipids and other highmolecular weight compounds that make the ex-traction of polyphenols difficult.

The extracts obtained in the first and secondcollector were analysed by rancimat method andall of them presented a protection factor loweror equal to one, perhaps due to the catalyticeffect of chlorophyll, which has a pro-oxidanteffect.

It is assumed that we are at optimum extrac-tion condition when the best protection factorcurve of the supercritical solid residues (ASR)was obtained, and this is attributed for thelemon balm plant at conditions of 10 MPa, 308K and 4 h.

The results obtained in this work indicate thatthe supercritical extraction could be an effectiveway of concentrating the antioxidant in solidmaterials at low pressures.


Acknowledgements

This work was sponsored by PAMAF throughproject D068-AROMED.

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melissa officinalis, l.: study of antioxidant activity in supercritical residues

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