SUPERCRITICAL FRACTIONATION OF EXTRACTS FROM ANNATTO SEEDS USING A HOME-MADE EQUIPMENT

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1 SUPERCRITICAL FRACTIONATION OF EXTRACTS FROM ANNATTO SEEDS USING A HOME-MADE EQUIPMENT J.C. J. Flores, A.L. Santana, M.A.A. Meireles LASEFI-School of Food Engineering University of Campinas, R. Monteiro Lobato, 80 CEP: Campinas SP Brazil, Telephone: 55 (19) juliojohner@gmail.com, adina.santana@gmail.com, and maameireles@gmail.com. ABSTRACT The goal of this work is the fractionation of annatto (Bixa orellana L.) seeds extracts using supercritical carbon dioxide, performed in a home-made system, constituted of a 100 ml extractor and two 90 ml separators. Annatto extracts were obtained at 313 K and 200 bar. Afterwards the extract was fractionated in the first separator at 312 K and 90 bar and 282 K and 40 bar in the second separator. The chemical profile of the extracts was analyzed using thin-layer chromatography using chloroform, ethanol and glacial acetic acid (95:05:01, v/v) as mobile phase and approximately 1µL for each sample was injected into TLC silica plates and compared to the bixin standard. The yield of extract was 2%, a 58% fraction was retained on the first separator and 42% on the second. The extracts presented distinct colorations and different compositions. KEYWORDS: supercritical fluid extraction, homemade extraction unit, annatto fractionation. 1. INTRODUCTION The plant annatto is indigenous to Brazil and is widely used as natural colorant due to its contents of carotenoids (Agner et al., 2004, Mercadante and Pfander, 2001, Russomanno et al., 2012). Supercritical fluid extraction (SFE) is an option to obtain high-value products in terms of healthpromoting properties (Cavalcanti et al., 2016). Some kinds of compounds from extracts obtained via SFE may present high cost and often are present in low concentration, which reduces its value for marketing as crude extract (Meireles, 2008). The separation of these extracts for obtaining of new products with distinct compositions is a way to improve the quality of extracts in terms of the high concentration of the compounds of interest (Wüst Zibetti et al., 2013, Gañán et al., 2015). Fractionation of natural extracts with supercritical fluids using the combination of separators result in the acquisition of high-quality products with increased purity and differentiation of bioactive compounds (Torres et al., 2016, Romero de la Vega et al., 2016). One of the difficulties in the extract fractionation is related to the solubility, i.e., the ability to separate volatile oils from other compounds that CO 2 can dissolve (Reverchon, 1997). The main factors that interfere in the separation process are pressure and temperature. At low temperatures (-5 up to +5 C) and waxes are insolubles in CO 2 (Reverchon and De Marco, 2006). Our challenge is to obtain SFE annatto extracts using a home-made system with a 100ml extractor, and subsequently fractionate these extracts using the same unit added of two 90 ml separators.

2 2. MATERIAL AND METHODS 2.1 Raw Material Annatto variety Piave was obtained from IAC (Agronomic Institute of Campinas), Department of Agriculture and Supply from the State of São Paulo, Brazil Supercritical Fluid Extraction The experimental assays of extraction, followed by subsequent fractionation were carried out in the home-made SFE-0.1L unit (Figure 1), built and validated elsewhere (Johner and Meireles, 2016). Carbon dioxide 99% pure (White Martins, Campinas, Brazil) was used as solvent and SFE assays were performed at 40 o C and 200 bar (Albuquerque and Meireles, 2012). The S/F=21 was used. The S/F ratio is the quantity of solvent necessary to exhaust the whole annatto seeds, and is calculated as the ratio between the mass of solvent and the mass of solute, in dry basis. Figure 1 - SFE-0.1L unit, the home-made equipment built in and validated in Lasefi (Johner and Meireles, 2016). After SFE step the tubing was connected directly to the first separator which pressure was maintained at 90 bar with the aid of the back pressure valve and the second separator was maintained at 40 bar, also regulated by the same type of valve. The first separator has a heating jacket which fluid circulates to maintain the temperature at the geometric center of the part at 39 C and the second separator had a fluid cooling jacket to maintain the temperature at the geometric center of the piece at 9 ºC. The pressurization step prevents losses of extracts inside the second separator or mixing of extract from the first separator. The extractor was depressurized through controlling of the micrometering valve in a proportion 10% lower than that used for the SFE. At first, the extractor was pressurized until reaching the same pressure as the first separator. Afterwards the first separator was depressurized until reaching the same pressure as the second one (Johner and Meireles, 2016). Back pressure valve was used in this work to control the outlet flow of solvent, although this type of valve is used to control pressure. The back pressure using procedure was carried out following steps of 10º twist paused for approximately 10 seconds.

3 After equalizing the pressures, the fractionated extracts were collected and the last back pressure valve was opened, performing the procedure previously mentioned. 2.3 Thin-Layer Chromatography Silica gel plates with aluminum backs (Alugram, Xtra SIL G, Macherey-Nagel, Germany) were used as stationary phase. The mobile was composed by chloroform (Merck, Darmstadt, Germany), ethanol (Chemco, Hortolandia, Brazil) and glacial acetic acid (Synth, Diadema, Brazil), in the proportion of 95:05:01, v/v (Wagner and Bladt, 2001). Approximately 10 μl of the samples were spotted on the thin-layer chromatography (TLC) plates with the aid of chromatographic syringe (Hamilton, Darmstadt, Germany) with approximately 1 cm distance from each band. Bixin standard was obtained previously by maceration of whole annatto seeds and subsequent exhaustive extraction with acetone for 6h (Johner and Meireles, 2016). ImageJ software (Ferreira and Rasband, 2010) was used to analyze the images obtained from the TLC plates in order to calculate the retardation factor (R F) and to quantify the compounds of interest, through comparison of the intensity in pixels, according to the methodology proposed elsewhere (Johner and Meireles, 2016). 3. RESULTS AND DISCUSSION Global yield of 2%, obtained from a S/F=21, was lower to that 3.8% obtained previously from a S/F=37 (Johner and Meireles, 2016), because higher values of S/F values implies in the exhausting the raw material, and consequently, higher extract yields. Besides that, lower yields are associated to the extract losses inside the separators (Figure 2C and 2D) and this occurred because the extracts are collected directly with the aid of the micrometering valve, which reduces the course of the extract until the flask collector. Global yield of extract from the first separator (Figure 2.A) is equivalent to 58% from the whole extract and that from the second separator (Figure 2.B) is equivalent to 41%. Depressurization step prevented the loss of extract in the pipeline located after the second separator. The use of cooling bath for the circulation of ethylene glycol solution to the second separator is another fact that allowed complete separation of extract from solvent. Obtaining of CO 2 with low residual content of extract is a determining factor for the recycling operation of this solvent., since the separation step for complete CO 2 can carry part of the extract to the recycling step which causes yield losses and damages the recycling equipment.

4 Figure 2 Extracts after fractionation. A First separator B - Second separator, C - First separator cover and D - First separator inside part. Bixin content on SFE extracts tends to increase as a function of S/F (Albuquerque et al., 2015). Using S/F=21 was observed that at the retardation factor (R F) of 0.55 that the extract from the first separator the intensity of bixin was higher than the extract obtained from the second one (Figure 3). Besides this, the intensity from both of extracts were lower than the intensity from the bixin standard, according to the methodology reported elsewhere (Johner and Meireles, 2016) that proposed a methodology to determine the concentration of bixin according to the intensity of pixels. At R F=0.87, both extracts presented positive answer with respect to the intensity of pixel, however with different concentrations and at R F=0.94 only the extract from the first separator presented intensity of pixels.

5 Figure 3 - Intensity of pixels from TLC plate of the fractionated extracts compared to the bixin standard. Evaluation of intensity in pixels from the TLC plate favored the distinction between the bands of compounds from each extract according to the R F values from 0.87 until According to the TLC plate (Figure 3) annatto fractionated extracts do not evidence relevance to the presence of bixin, but other group of compounds. Therefore, the utilization of other substances as standards and of spray reagents support the identification and quantification of other types of compounds. 4. CONCLUSION Improvement of annatto extracts fractionation is attributed to the control precision over the differential of pressure between the separators during their collection. Fractionation of annatto extracts resulted on two different extracts with relevant profile of bioactive compounds, attributing to these products great potential for commercial uses for food, drugs and cosmetics. 5. ACKNOWLEDGEMENTS The authors acknowledge CAPES (2952/2011), CNPq (302423/2015-0), CNPq (140287/2013-2) and FAPESP (RT2015/ ) for financial assistantship. 6. REFERENCES Agner, A. R., Barbisan, L. F., Scolastici, C. & Salvadori, D. M Absence of carcinogenic and anticarcinogenic effects of annatto in the rat liver medium-term assay. Food Chem Toxicol, 42, (10), Albuquerque, C. L. C. & Meireles, M. A. A Defatting of annatto seeds using supercritical carbon dioxide as a pretreatment for the production of bixin: Experimental,

6 modeling and economic evaluation of the process. The Journal of Supercritical Fluids, 66, Albuquerque, C. L. C., Santana, Á. L. & Meireles, M. A. A Thin Layer Chromatographic Analysis of Annatto Extracts Obtained Using Supercritical Fluid. Food and Public Health, 5, (4), Cavalcanti, R. N., Albuquerque, C. L. C. & Meireles, M. A. A Supercritical CO2 extraction of cupuassu butter from defatted seed residue: Experimental data, mathematical modeling and cost of manufacturing. Food and Bioproducts Processing, 97, Ferreira, T. & Rasband, W The ImageJ User Guide IJ [Online]. Available: imagej.nih.gov/ij/docs/guide/. Gañán, N. A., Dambolena, J. S., Martini, R. E. & Bottini, S. B Supercritical carbon dioxide fractionation of peppermint oil with low menthol content Experimental study and simulation analysis for the recovery of piperitenone. The Journal of Supercritical Fluids, 98, Johner, J. C. F. & Meireles, M. A. A Construction of a supercritical fluid extraction (SFE) equipment: validation using annatto and fennel and extract analysis by thin layer chromatography coupled to image. Food Science & Technology, Meireles, M. A. A Supercritical Extraction Plants Equipment, Process, and Costs. In: Martinez, J. L. (ed.) Supercritical fluid extraction of nutraceuticals and bioactive compounds. London New York: CRC Press Taylor & Francis Group. Mercadante, A. Z. & Pfander, H Caracterização de um novo carotenóide minoritário de urucum. Ciênc. Tecnol. Aliment., Campinas, 21, (2), Reverchon, E Supercritical fluid extraction and fractionation of essential oils and related products. The Journal of Supercritical Fluids, 10, (1), Reverchon, E. & De Marco, I Supercritical fluid extraction and fractionation of natural matter. The Journal of Supercritical Fluids, 38, (2), Romero de la Vega, G., Salgado Cervantes, M. A., Garcia Alvarado, M. A., Romero- Martínez, A. & Hegel, P. E Fractionation of vanilla oleoresin by supercritical CO2 technology. The Journal of Supercritical Fluids, 108, Russomanno, O. M. R., Kruppa, P. C. & Fabri, E. G DOENÇAS FÚNGICAS DO URUCUM. Biológico, 74, (1), Torres, R. A. C., Santana, Á. L., Santos, D. T. & Meireles, M. A. A Perspectives on the Application of Supercritical Antisolvent Fractionation Process for the Purification of Plant Extracts: Effects of Operating Parameters and Patent Survey. Recent Patents on Engineering, 10, (2), Wagner, H. & Bladt, S Plant Drug Analysis: A Thin Layer Chromatography, Germany. Wüst Zibetti, A., Aydi, A., Arauco Livia, M., Bolzan, A. & Barth, D Solvent extraction and purification of rosmarinic acid from supercritical fluid extraction fractionation waste: Economic evaluation and scale-up. The Journal of Supercritical Fluids, 83,