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Rapid Techniques for the Extraction of Vitamin E Isomers from Amaranthus caudatus Seeds: Ultrasonic and Supercritical Fluid Extraction Renato Bruni, 1 Alessandra Guerrini, 1 Santo Scalia, 2 Carlo Romagnoli 3 and Gianni Sacchetti 4 * 1 Department of Chemistry, University of Ferrara, Via Luigi Borsari 42, I-44100 Ferrara, Italy 2 Department of Pharmaceutical Sciences, University of Ferrara, Via Fossato di Mortara 17-19, I-44100 Ferrara, Italy 3 Department of Animal Biology, Section of Botanical Garden, University of Modena and Reggio Emilia, v. le Caduti in Guerra 127, I-41100 Modena, Italy 4 Department of Biology, Pharmaceutical Biology Lab., C. so Porta Mare 2, I-44100 Ferrara, Italy Supercritical fluid extraction (SFE) of seeds of Amaranthus caudatus (Amaranthaceae) and the use of ultrasound as a co-adjuvant in the extraction process were compared with methods traditionally used in the extraction of tocopherols and fatty acids. The use of readily available ultrasound equipment as an adjunct to the classical methods employed for the extraction of tocols provided qualitatively acceptable results more rapidly and more economically. SFE gave quantitatively better yields in shorter times, with solvent-free extracts obtained under conditions that minimised the degradation of thermolabile components. No significant variations were observed in the profile of the fatty acids extracted from amaranth oil by SFE or other methods, thus confirming the qualitative comparability of the faster supercritical extraction with the more time-consuming classical techniques even when processed with the aid of ultrasound. Copyright # 2002 John Wiley & Sons, Ltd. Keywords: Supercritical fluid extraction; ultrasonic extraction; vitamin E isomers; tocopherols; fatty acids; amaranth oil; Amaranthus caudatus. INTRODUCTION Vitamin E isomers are important natural anti-oxidants, widely employed in cosmetics and in health foods, but they often prove difficult to extract because they are sensitive to temperature and oxidation. The classic procedures used to extract and isolate non-saponifiable components have distinct drawbacks in that they are time-consuming and labour-intensive, they may subject the source material to severe conditions that can damage the most sensitive components, they necessitate the handling of large volumes of hazardous solvents, and they involve extended concentration steps which can result in the loss or degradation of target analytes (Bevan and Marshall, 1994). Such conditions accentuate the attention currently being focused on alternative extrac- tion methods which provide rapid, efficient and economic extraction under mild conditions with a limited environ- mental impact. Several technologies have been described to obtain the tocopherol fraction from plant material using both traditional solvents and supercritical fluids. Of these, supercritical fluid extraction (SFE) has been the object of numerous studies aimed at optimising the extraction conditions for lipophilic compounds, including tocols, and evaluating the use of polar modifiers to extract any polar components present in the various matrices, including plant material (Bevan and Marshall, 1994). The use of supercritical-phase carbon dioxide and low temperatures not only reduces the impact on the environment but also ensures efficiency and repeatability as well as a significant decrease in operating costs and times (Bartle, 1990). Although the use of ultrasound as a co-adjuvant in the extraction process has been the subject of many environmental studies (Babic et al., 1998; Lombas-Garcia et al., 1998), the methodology has not enjoyed the same attention in the field of plant matrices (Sargenti and Vichnewski, 2000). Grain amaranth (Amaranthus spp.; Amaranthaceae) is a broadleaf pseudo-cereal native to the plateaus of South America where it has long been used as a source of food (Sauer, 1967; Williams, 1995). Various reports have described its potential as a new crop (Kauffman and Weber, 1990; Myers, 1996; Mapes et al., 1997) owing primarily to its high protein content (12–17%), the available fibre, and the presence of lysine and tocopherol (Becker, 1989; Chaturvedi et al., 1993). In addition, the food value of amaranth seed is enhanced by its fatty acid profile which is similar to that of corn oil and which is often correlated with the tocopherol content, since the tocopherols are known to protect fatty acids (Lyon and Becker, 1987; Johns and Romeo, 1997). Given the ability of amaranth to adapt to unfavourable climatic conditions present in various areas of the developing world (Cheeke et al., 1981), this grain has recently been rediscovered, and its reintroduction as a food source has been widely supported to compensate for the scarcity of other grains (Mujica, 1994) and to make up for local nutritional deficiencies. However, the characteristics described above also make Amaranthus spp. an inter- esting crop for the Western market, especially as a health PHYTOCHEMICAL ANALYSIS Phytochem. Anal. 13, 257–261 (2002) Published online in Wiley InterScience (www.interscience.wiley.com). DOI: 10.1002/pca.651 Copyright # 2002 John Wiley & Sons, Ltd. * Correspondence to: G. Sacchetti, Department of Biology, Pharmaceutical Biology Laboratory, C. so Porta Mare 2, I-44100 Ferrara, Italy. Email: [email protected] Received 20 February 2001 Revised 13 September 2001 Accepted 14 September 2001

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Page 1: Rapid techniques for the extraction of vitamin E isomers from Amaranthus caudatus seeds: ultrasonic and supercritical fluid extraction

Rapid Techniques for the Extraction of VitaminE Isomers from Amaranthus caudatus Seeds:Ultrasonic and Supercritical Fluid Extraction

Renato Bruni,1 Alessandra Guerrini,1 Santo Scalia,2 Carlo Romagnoli3 and Gianni Sacchetti4*1Department of Chemistry, University of Ferrara, Via Luigi Borsari 42, I-44100 Ferrara, Italy2Department of Pharmaceutical Sciences, University of Ferrara, Via Fossato di Mortara 17-19, I-44100 Ferrara, Italy3Department of Animal Biology, Section of Botanical Garden, University of Modena and Reggio Emilia, v. le Caduti in Guerra 127,I-41100 Modena, Italy4Department of Biology, Pharmaceutical Biology Lab., C. so Porta Mare 2, I-44100 Ferrara, Italy

Supercritical fluid extraction (SFE) of seeds of Amaranthus caudatus (Amaranthaceae) and the use ofultrasound as a co-adjuvant in the extraction process were compared with methods traditionally used in theextraction of tocopherols and fatty acids. The use of readily available ultrasound equipment as an adjunct tothe classical methods employed for the extraction of tocols provided qualitatively acceptable results morerapidly and more economically. SFE gave quantitatively better yields in shorter times, with solvent-freeextracts obtained under conditions that minimised the degradation of thermolabile components. Nosignificant variations were observed in the profile of the fatty acids extracted from amaranth oil by SFE orother methods, thus confirming the qualitative comparability of the faster supercritical extraction with themore time-consuming classical techniques even when processed with the aid of ultrasound. Copyright� 2002 John Wiley & Sons, Ltd.Keywords: Supercritical fluid extraction; ultrasonic extraction; vitamin E isomers; tocopherols; fatty acids; amaranth oil;Amaranthus caudatus.

INTRODUCTION

Vitamin E isomers are important natural anti-oxidants,widely employed in cosmetics and in health foods, butthey often prove difficult to extract because they aresensitive to temperature and oxidation. The classicprocedures used to extract and isolate non-saponifiablecomponents have distinct drawbacks in that they aretime-consuming and labour-intensive, they may subjectthe source material to severe conditions that can damagethe most sensitive components, they necessitate thehandling of large volumes of hazardous solvents, andthey involve extended concentration steps which canresult in the loss or degradation of target analytes (Bevanand Marshall, 1994). Such conditions accentuate theattention currently being focused on alternative extrac-tion methods which provide rapid, efficient and economicextraction under mild conditions with a limited environ-mental impact.

Several technologies have been described to obtain thetocopherol fraction from plant material using bothtraditional solvents and supercritical fluids. Of these,supercritical fluid extraction (SFE) has been the object ofnumerous studies aimed at optimising the extractionconditions for lipophilic compounds, including tocols,and evaluating the use of polar modifiers to extract anypolar components present in the various matrices,including plant material (Bevan and Marshall, 1994).

The use of supercritical-phase carbon dioxide and lowtemperatures not only reduces the impact on theenvironment but also ensures efficiency and repeatabilityas well as a significant decrease in operating costs andtimes (Bartle, 1990). Although the use of ultrasound as aco-adjuvant in the extraction process has been the subjectof many environmental studies (Babic et al., 1998;Lombas-Garcia et al., 1998), the methodology has notenjoyed the same attention in the field of plant matrices(Sargenti and Vichnewski, 2000).

Grain amaranth (Amaranthus spp.; Amaranthaceae) isa broadleaf pseudo-cereal native to the plateaus of SouthAmerica where it has long been used as a source of food(Sauer, 1967; Williams, 1995). Various reports havedescribed its potential as a new crop (Kauffman andWeber, 1990; Myers, 1996; Mapes et al., 1997) owingprimarily to its high protein content (12–17%), theavailable fibre, and the presence of lysine and tocopherol(Becker, 1989; Chaturvedi et al., 1993). In addition, thefood value of amaranth seed is enhanced by its fatty acidprofile which is similar to that of corn oil and which isoften correlated with the tocopherol content, since thetocopherols are known to protect fatty acids (Lyon andBecker, 1987; Johns and Romeo, 1997). Given theability of amaranth to adapt to unfavourable climaticconditions present in various areas of the developingworld (Cheeke et al., 1981), this grain has recently beenrediscovered, and its reintroduction as a food source hasbeen widely supported to compensate for the scarcity ofother grains (Mujica, 1994) and to make up for localnutritional deficiencies. However, the characteristicsdescribed above also make Amaranthus spp. an inter-esting crop for the Western market, especially as a health

PHYTOCHEMICAL ANALYSISPhytochem. Anal. 13, 257–261 (2002)Published online in Wiley InterScience (www.interscience.wiley.com). DOI: 10.1002/pca.651

Copyright � 2002 John Wiley & Sons, Ltd.

* Correspondence to: G. Sacchetti, Department of Biology, PharmaceuticalBiology Laboratory, C. so Porta Mare 2, I-44100 Ferrara, Italy.Email: [email protected]

Received 20 February 2001Revised 13 September 2001

Accepted 14 September 2001

Page 2: Rapid techniques for the extraction of vitamin E isomers from Amaranthus caudatus seeds: ultrasonic and supercritical fluid extraction

food and as a source of tocopherols for the cosmeticsindustry.

In the present paper, SFE and the use of ultrasound, asa co-adjuvant in the extraction process, are comparedwith other methods traditionally used in the extraction oftocopherols and fatty acids from seeds of Amaranthuscaudatus.

EXPERIMENTAL

Plant material

Commercial seed material of Amaranthus caudatus wasground using a blade grinder and forced through a0.2 mm mesh sieve, care being taken to ensure that duringthe processing the temperature never exceeded 30°C.After grinding, the flour was stored in the dark at �20°C.

Extraction of tocopherol

Three extraction methods were evaluated with respect tothe amount of tocopherols that could be extracted fromsamples of seed flour of A. caudatus, namely, classicalextraction using organic solvents (methanol and hexane),ultrasound-enhanced extraction and SFE. The sameamount of sample was used for each method and carewas taken to protect the operations from light and fromoxidising conditions. Each extraction was repeated threetimes. The extracts, obtained according to the variousmethods described below, were stored in the dark at�20°C until required for quantitative HPLC analysis.

Method 1. Seed flour of A. caudatus (5 g) was placed in aflask containing 100 mL of methanol and stirred for 24 hin the dark at a constant temperature of 25°C. Thesamples were then filtered and centrifuged for 20 min at3000 g and the supernatant dried in a rotary evaporatorand weighed. The dry extract was covered with 100 mLof hexane and placed in a Branson (Danbury, CT, USA)model 5200 ultrasound bath for 30 min at a constanttemperature of 25°C to facilitate solubilisation. Subse-quently, the sample was centrifuged at 3000 g for 20 min,and the supernatant was dried using a rotary evaporatorand stored at �20°C until required for analysis.

Method 2. Seed flour of A. caudatus (5 g), suspended in100 mL of methanol, was subject to ultrasound treatmentfor 1 h in the dark at a constant temperature of 25°C. Theextract obtained was then centrifuged at 3000 g for20 min and treated as indicated above.

Method 3. Seed flour of A. caudatus (5 g) was subjectedto supercritical carbon dioxide extraction using anApplied Separations (Allentown, PA, USA) model Spe-ed SFE extractor which comprised an air-driven pump todeliver the carbon dioxide to the extraction cell (a 10 mLstainless steel vessel with 2 �m frits at either end) housedwithin a temperature-controlled oven. The outlet of theextraction cell was connected to a thermally controlledvariable restrictor which maintained supercritical pres-sure conditions in the system. The SFE instrument andextraction vessel were rated to withstand pressures of upto 400 atm.

SFE extractions were performed on flour samples

under the following operating conditions: carbon dioxideflow-rate, 2 L/min; oven temperature, 40°C; and restric-tor temperature, 70°C. One sample was extracted at apressure of 200 atm, whilst a second sample wasextracted at 400 atm: for both extractions, a 1 min staticextraction was followed by 14 min of dynamic extrac-tion. As the carbon dioxide evaporated at the restrictoroutlet due to decompression, the extracted material wascollected in a glass vial fitted with a septum and a needlevent. The extracted samples were stored in the dark at�20°C until required for analysis.

HPLC apparatus and chromatographic conditions

Analyses were performed using a Jasco (Tokyo, Japan)modular HPLC [consisting of a model PU-980 pump, anLG-1580-02 ternary gradient unit, a DG-980-50 three-line degaser, and a 975 UV–visible detector (set at awavelength of 295 nm)] linked to an injection valve witha 20 �L sampler loop. A Lichrosorb (Teknokroma,Barcelona, Spain) silica gel Si-60 column (25 � 0.4 cmi.d.; 5 �m) was used, and the mobile phase was 0.5%isopropanol in hexane at a flow-rate of 1 mL/min; theinjection volume was 40 �L. All solvents employed wereof chromatographic grade. The tocopherol peaks from theamaranth samples were identified by comparing theirspectra with spectra obtained using pure, authenticstandards of �-, �-, �- and �-tocopherol obtained fromMatreya (Pleasant Gap, PA, USA). Peak areas weredetermined by integration using dedicated software(Borwin version 1.22; JMBS Developments, Grenoble,France). The qualitative/quantitative analysis of eachextract was performed in triplicate.

Extraction of fatty acids

The fatty acid content was evaluated in extracts obtainedby SFE (method 3) and in extracts obtained using method2 but with hexane as the extracting solvent.

Gas chromatography of methyl esters of fatty acids

Fatty acid methyl esters were prepared by transmethyl-ation with sodium methoxide in the presence of methylacetate following the method of Christie (1982).Analyses were performed using a Fisons (Milano, Italy)model 9130 VIC 900 gas chromatograph fitted with anEL 980 processor, an FID detector, and a Mega(Legnano, Italy) SE52 column (25 m � 0.32 mm i.d.;0.1 �m film thickness). The analytical conditions were:injection temperature, 300°C; detector temperature,350°C; split ratio, 1:50; carrier gas, helium at a flow-rate of 2 mL/min; temperature programme, initially150°C rising at 5°C/min to 250°C. Authentic fatty acidstandards were obtained from Alltech (Deerfield, IL,USA).

RESULTS AND DISCUSSION

In terms of yield of raw extract, the extraction strategiesapplied to the flour of A. caudatus proved relativelyhomogeneous (Table 1) and gave results similar to thosepreviously reported (Lehmann et al., 1994; Budin et al.,1996). The lowest extraction capacity was exhibited by

258 R. BRUNI ET AL.

Copyright � 2002 John Wiley & Sons, Ltd. Phytochem. Anal. 13: 257–261 (2002)

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SFE at 200 atm. On the other hand, SFE yielded thehighest amounts of total tocopherols (Table 2), provingnearly twice as effective as extractions performed bysoaking the material in organic solvents for 24 h (method1) and by sonication treatment in an ultrasound bath(method 2). SFE at 400 atm gave a total yield oftocopherol some 20% higher than that obtained at 200atm.

The extraction yields obtained in this study appearmuch higher than those found in the literature for thesame plant matrix, thus confirming the high biodiversityof this species with respect to the class of compoundsstudied (Imeri et al., 1987). The extraction methods used,however, provided homogeneous, quantitative data. Themost significant variability was shown by �-tocopherol,which was quite abundant in all extracts with the lowestamounts being obtained using ultrasound. The variationin amounts found for both total and individual tocopher-ols may be explained by the fact that in SFE the matrix–solvent interactions differ from those present in tradi-tional organic solvent extraction (Bartle, 1990) and thismay differently affect how the tocopherol solubilises inthe extraction solvent. Moreover, the absence of contactwith atmospheric oxygen during the extraction processmakes it possible to limit the breakdown of tocopherol.

In the HPLC analysis of the samples, �-, �- and�-tocopherol were well separated and could be clearlyidentified (Fig. 1); on the other hand, while �-tocopherolcould also be clearly identified by comparison with purestandards [Fig. 1(I)], in chromatograms of samples [Fig.1(II–IV)] a large adjacent peak appeared which was mostlikely due to �-tocotrienol. In addition, the chromato-grams of samples extracted using SFE [Fig. 1(IV)]showed a peak between �- and �-tocopherol [most likelydue to the presence of �- or �-tocotrienol (Lehmann et al.,

1994; Kamal-Eldin et al., 2000)], and also showed anunidentified peak between �- and �-tocopherol. For thisreason, SFE proved less selective than ultrasound in theextraction of tocopherols alone, although the qualitativeand quantitative results were markedly better.

Amaranth oil extracted using the different methodsdescribed showed no significant variations in fatty acidcomposition and all the values obtained with the differentextraction methods fell within the range described in the

Table 1. Yield of crude extract obtained from seeds ofAmaranthus caudatus using various extractionmethods

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Table 2. Total tocopherol content and content of �-, �-, �- and �-tocopherol in seeds of Amaranthus caudatus determinedfollowing various methods of extraction

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EXTRACTION OF VITAMIN E ISOMERS FROM A. CAUDATUS 259

Copyright � 2002 John Wiley & Sons, Ltd. Phytochem. Anal. 13: 257–261 (2002)

Page 4: Rapid techniques for the extraction of vitamin E isomers from Amaranthus caudatus seeds: ultrasonic and supercritical fluid extraction

literature (Lehmann et al., 1994; Budin et al., 1996), thusconfirming the qualitative comparability of the faster SFEcompared with the more time consuming classicaltechnique. Under the various SFE extraction conditions,the proportion of fatty acids varied between 16.6 and19.9% for palmitic acid (C16), and 25.5 and 26.8% foroleic acid (C18:1), with similar results being obtainedwith ultrasound-assisted hexane extraction (Table 3). Forlinoleic acid (C18:2), the unsaturated fatty acid ofgreatest interest for food (Beliz and Grosch, 1999), theextract obtained using hexane in an ultrasound bathcontained a slightly higher level (53.0%) than did SFE-produced extracts (51.6 and 52.2% at 200 and 400 atm,respectively). On the other hand, SFE at 400 atm wasmore efficient at extracting the minor, longer-chain fattyacids than SFE at 200 atm and the classical extraction,which both showed similar results. This was the mostinteresting result from the comparison of extractionmethods, since SFE at 400 atm provided a moreexhaustive extraction of the fatty acids than didtraditional organic solvent extraction, even with the aidof ultrasound. Moreover such results were achieved morequickly and using less organic solvents.

A comparison with traditional extraction shows thatthe use of ultrasound as an adjunct to the classicalextraction of tocols provides qualitatively acceptable

results but much more quickly, more economically andusing equipment commonly available in the laboratory.Its use can, therefore, be considered for the qualitativescreening of a large number of samples. Such comparisonalso clearly shows that supercritical fluid extractionprovides quantitatively better results in shorter times and,being under conditions that prevent contact with atmos-pheric oxygen, with minimal degradation of thermolabilecomponents. Moreover, the extracts obtained are solvent-free. The advantages and inherent potential of super-critical fluid extraction are well known and have beenwidely described in the literature (Bartle, 1990). SFE,furthermore, permits easier scale-up from analytical tosemi-preparative conditions, thus circumventing the costsinvolved in the purchase and disposal of the solventsused. The fact that SFE can yield plant extracts whichhave never come into contact with conventional organicsolvent, and thus can be directly used “as is” by the foodand cosmetics industries, makes this technique a veryattractive alternative to the methods currently in use.

Acknowledgements

The authors wish to thank Eileen N. Cartoon for the English translationof this manuscript.

REFERENCES

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Table 3. Fatty acid content of seeds of Amaranthus caudatus determined following various methods of extraction

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260 R. BRUNI ET AL.

Copyright � 2002 John Wiley & Sons, Ltd. Phytochem. Anal. 13: 257–261 (2002)

Page 5: Rapid techniques for the extraction of vitamin E isomers from Amaranthus caudatus seeds: ultrasonic and supercritical fluid extraction

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EXTRACTION OF VITAMIN E ISOMERS FROM A. CAUDATUS 261

Copyright � 2002 John Wiley & Sons, Ltd. Phytochem. Anal. 13: 257–261 (2002)