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Optimization of rutin isolation from Amaranthus paniculatus leaves by high pressure extraction and fractionation techniques
Accepted Manuscript Title: Optimization of rutin isolation from Amaranthus paniculatus leaves by high pressure extraction and fractionation techniques Author: Paulius Kraujalis Petras Rimantas Venskutonis Elena Ib´an˜ ez Miguel Herrero PII: DOI: Reference:
S0896-8446(15)30049-8 http://dx.doi.org/doi:10.1016/j.supflu.2015.06.022 SUPFLU 3370
To appear in:
J. of Supercritical Fluids
Received date: Revised date: Accepted date:
28-3-2015 25-6-2015 25-6-2015
Please cite this article as: Paulius Kraujalis, Petras Rimantas Venskutonis, Elena Ib´an˜ ez, Miguel Herrero, Optimization of rutin isolation from Amaranthus paniculatus leaves by high pressure extraction and fractionation techniques, The Journal of Supercritical Fluids http://dx.doi.org/10.1016/j.supflu.2015.06.022 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
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Optimization of rutin isolation from Amaranthus paniculatus leaves by high
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pressure extraction and fractionation techniques
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Paulius Kraujalisa, Petras Rimantas Venskutonisa, Elena Ibáñezb*, Miguel Herrerob
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a
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[email protected]
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TEL: +34 910 017 956
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FAX: +34 910 017 905
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Graphical abstract
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Highlights
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70/30 (v/v) water/ethanol ratio, at 188°C and 20 min were optimal parameters for
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Kaunas University of Technology, Department of Food Science and Technology, Radvilėnų pl. 19, LT-50254, Kaunas, LITHUANIA b
Foodomics Laboratory, Institute of Food Science Research (CIAL, CSIC), Nicolás Cabrera 9, Campus UAM Cantoblanco,28049 - Madrid, SPAIN * Corresponding Author (Elena Ibáñez)
rutin recovery.
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PLE recovered 4 times more of rutin than conventional extraction.
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Application of SAF increased rutin concentration up to 1.5 times.
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A PLE-SAF integrated process was successfully optimized and applied.
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Abstract
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A procedure based on the application of pressurized liquid extraction (PLE) was
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employed to recover the major Amaranthus spp. flavonoid glycoside rutin from dried
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ground plant leaves. A central composite design (CCD) was used to optimize rutin
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extraction; three factors were considered: extraction solvent (water/ethanol ratio, from
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0/100 to 100/0), extraction time (from 5 to 20 min) and extraction temperature (from 50
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to 200°C). Optimization of the three extraction parameters revealed that water/ethanol
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ratio in the solvent mixture was the most-significant factor influencing rutin yield,
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followed by extraction temperature. The total rutin recovery varied from 6.95 to 14.03
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g/kg dry matter (DM) depending on the experimental conditions tested. The optimum
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extraction conditions for the highest rutin content were 70/30 (v/v) water/ethanol ratio,
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at 188°C and 20 min as static extraction time. In this case rutin recovery from the leaves
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(14.30 g/kg DM) was 4-times higher compared to that attainable using conventional
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ultrasound assisted extraction (3.62 g/kg DM) with methanol:ethanol mixture (90:10) as
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solvent. Rutin recovery from defatted and non-defatted seeds of amaranth under
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optimum PLE conditions was 35.3 and 41.1 mg/kg DM, respectively, whereas using
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ultrasound assisted extraction 19.7 and 34.6 mg/kg DM were obtained, respectively.
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Extracts obtained under optimum conditions were further fractionated by using
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supercritical antisolvent fractionation (SAF). Extracts of amaranth leaves containing
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70% water were mixed with supercritical CO2 and separated into two fractions
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according to the molecular mass and solubility of the contained compounds. A central
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composite experimental design was applied for the determination of the best
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fractionation conditions to maximize rutin enrichment. Maximum rutin concentration in
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raffinate (22.57 g/kg) was recovered at 15 MPa using a feed mixture flow rate of 0.3
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mL/min.
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Keywords: amaranth, rutin, pressurized liquid extraction, optimization,
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supercritical antisolvent fractionation
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3
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1. Introduction
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Amaranth (Amaranthus spp.) is a multipurpose valuable crop supplying nutritional
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grains, leafy vegetables for food and animal feed, as well as medicinal herb [1].
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Amaranth seeds oil has a high content of unsaturated fatty acids, triterpene squalene [2]
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and
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Glutein-free amaranth seeds are the source of high quality protein consisting of easily
60
digestible albumins and globulins, which are the main components of highly nutritive
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amaranth proteins [4]. In comparison with fine wheat flour, whole amaranth flour has
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high lysine content [5].
natural lipophilic tocopherol antioxidants possessing vitamin E activity [3].
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Leafy parts of amaranth contain various constituents which possess important effects
64
on processing properties and human health. Green leafy vegetables of amaranth are a
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rich source of dietary fibers and others functional components [6] such as phenolic
66
acids, flavonoids and their glycosides. In this regard, rutin (quercetin-3-O-rutinoside,
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Figure 1) is the main glycoside found in all anatomical parts of amaranth, although
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leaves and flowers contain the highest amount of rutin [7]. The specific content of rutin
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in amaranth depends on species, variety and growing conditions, among other factors
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[8].
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Rutin is a flavonoid found in many plants including buckwheat, kacip fatimah,
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prayer plant, Flos Sophorae Immaturus, tobacco leaves, etc. [9-13]. Numerous studies
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have reported different biological and pharmacological properties of rutin either in vivo
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or in vitro as well as a risk reduction effect on diverse diseases, thus, promoting health
75
[14]. In fact, rutin possesses several biological activities, including cytoprotective,
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antiplatelet, anti-inflammatory, anti-tumor, and antibacterial activities [15-20]. Due to
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these positive activities, numerous extraction methods have been developed to obtain
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rutin from various plant materials. The techniques employed range from conventional
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solid-liquid extraction (SLE) to more sophisticated advanced processes such as
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supercritical fluid extraction (SFE) [14]. Pressurised liquid extraction (PLE) and
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subcritical water extraction (SWE) are promising extraction techniques that make use of
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elevated temperature and pressure to improve the efficiency of the extraction procedure,
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shortening the extraction time and reducing solvent consumption [21]. On the other
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hand, supercritical fluids (SCF) are alternative solvents which could be used for
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advanced separation techniques in different processes. Recently, supercritical
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antisolvent fractionation (SAF) has been suggested as an interesting alternative to
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fractionate phenolic compounds from plant extracts taking advantage of the differences
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in solubility of these compounds in the solvent and in supercritical CO2 [22]. SAF is
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achieved by the continuous contact between the supercritical carbon dioxide (SC-CO2)
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and a polar liquid mixture at pressurized conditions [23]. Under these conditions, the
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SCF is able to dissolve the less polar solvent and the non-polar compounds in the
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mixture SC-CO2 + solvent, selectively precipitating heavier, more polar compounds that
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are not soluble in the fractionation medium [24]. Considering the large number of
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factors involved in the above mentioned techniques, the use of experimental designs to
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optimize the process conditions is recommended. Response surface methodology
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(RSM) applied through a central composite experimental design (CCD) is commonly
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used to evaluate the effects and interactions of multiple factors and is useful for building
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second order model for response variables [2, 25, 26].
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Thus, the goal of the present study was to develop an integrated process, firstly by
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optimizing the PLE conditions (solvent ratio, extraction time and temperature) to
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maximize the rutin extraction from amaranth leaves, and secondly, by optimizing the
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SAF conditions of the PLE extract (containing rutin and other plant components such as
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sugars and proteins) to obtain a fraction highly enriched in rutin. To the best of our
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knowledge, this is the first time when both green techniques (PLE + SAF) are being
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employed for rutin recovery from amaranth leaves.
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2. Materials and methods
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2.1. Samples and chemicals.
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Aerial parts of amaranth (Amaranthus paniculatus) were collected during flowering
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stage in 2013 in an organic herb farm (Panara village, Varėna district, Lithuania). The
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plants were dried at 40°C by active ventilation in the dark. Dry leaves of amaranth were
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ground in a Retsch GM 200 mill (Haan, Germany). Before grinding, stems were
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separated from leaves and not used for analysis.
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Extraction solvents used were ethanol, extra pure (Scharlau, Scharlab, Spain),
115
methanol HPLC grade (VWR, Radnor, Pennsylvania, USA), and water purified with a
116
Milli-Q system (Millipore, Bedford, MA, USA). For the chromatographic analysis,
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solvents used were of HPLC grade. Acetic acid (≥99%) and rutin hydrate (≥94 %,
118
HPLC) were obtained from Sigma Aldrich (Steinheim, Germany).
119 120
2.2. Pressurized liquid extraction (PLE)
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PLE was performed in an accelerated solvent extractor ASE-200 (Dionex,
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Sunnyvale, CA, USA) using 2 g of sample, which was mixed with washed sea sand
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(Panreac, Barcelona) (1:1) and placed in a 11 mL volume stainless-steel cell. Extraction
124
solvents (ethanol and MiliQ water) were sonicated before extraction to remove any
125
possible dissolved air. Extraction cells were equipped with stainless steel frits and
126
cellulose filters at both ends to avoid solid particles in the collection vial. Once the
127
extraction cell was loaded with sample, an instrumentally-preset heating-up step was
128
applied for a given time prior to any extraction. This warming up time changed
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depending on the extraction temperature (i.e., 5 min when the extraction temperature
130
ranged from 50 to 100 °C, and 6, 8 or 9 min if the extraction temperature was 124, 168
131
or 198 °C, respectively). All experimental runs were performed at 10 MPa and used one
132
static cycle. After each extraction, cells were flushed with 60% cell volume of fresh
133
solvent and finally purged 60 s with nitrogen. The extracts were kept at 4 °C until use
134
for analysis. Extraction time, temperature and solvent ratio were studied using a rotable
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central composite experimental design (see below) covering the entire instrument’s
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working range.
137 138
2.3. Supercritical antisolvent fractionation (SAF)
139
SAF was performed using a Helix supercritical fluid extractor (Applied
140
Separations, Allentown, PA, USA) with some modifications in order to introduce liquid
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feed into supercritical carbon dioxide (SC-CO2) flow (Figure 2). The extract obtained
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by PLE was mixed with SC-CO2 under constant flow and recovered into two fractions.
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Two pumps were used for the fractionation of liquid mixture: the extract after PLE
144
(liquid feed) was delivered by HPLC pump and CO2 by liquid CO2 pump. Liquid feed
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was mixed with SC-CO2 stream in the heated pipe just before the precipitation vessel
146
inlet and sprayed into the precipitation vessel through the nozzle to reach vapour-liquid
147
equilibrium. A precipitation vessel was used to separate the two phases in equilibrium;
148
the liquid phase (compounds insoluble in SC-CO2) collected at the bottom of
149
precipitation vessel, whereas SC-CO2 soluble compounds (vapour phase) exited through
150
the top of precipitation vessel and were recovered in the separator vessel. Twenty mL of
151
liquid feed was used for all fractionation experimental runs. The first fraction (raffinate)
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was recovered in the pressurized stainless steel precipitation vessel, while the second
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fraction (extract) was collected in a separator at atmospheric pressure. The pressure of
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154
SC-CO2 and the liquid feed flow rate were determined from a CCD (see below),
155
whereas the temperature of precipitation vessel and separator was constant at 40 °C and
156
30 °C, respectively. CO2 flow rate was kept constant (0.216 kg/h) during all
157
experimental runs. The attained raffinate was freeze-dried whereas the extract was dried
158
under a gentle nitrogen stream. Both fractions were placed in the refrigerator until
159
analysis.
160
Three components system composed by PLE extract (70% water and 30% ethanol)
161
and supercritical CO2 was employed for fractionation of rutin. The ternary system was
162
composed by a supercritical fluid and a polar mixture as well as the compounds that
163
have been fractionated. Optimization of the conditions to achieve the fractionation into
164
the liquid and the gaseous phase (raffinate and extract, respectively) was performed
165
selecting a zone in the ternary phase equilibria (SC-CO2-EtOH-H2O system) in which
166
the mixture could be separated apart. Further details on the mole fraction equilibrium
167
data for this ternary system at 40 ºC and pressures of 100-300 bar can be found
168
elsewhere (27). By analyzing this theoretical data, it can be seen that the ethanol
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partition coefficients are influenced by the pressure, the mass percentage of water in the
170
feed and the feed/SC-CO2 flow rate ratio. In the present study, pressure and feed/SC-
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CO2 flow rate (expressed as feed mixture flow rate) have been selected while % of
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water in the feed was fixed at 70%.
173 174
2.4. Experimental designs
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Response surface methodology (RSM) was applied twice, firstly to determine the
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optimum PLE conditions (solvent ratio, extraction time and temperature) to maxime
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extract yield and rutin recovery, and secondly, to optimize the SAF conditions to
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fractionate the PLE extract to obtain a new extract highly enriched in rutin. For this
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purpose statistical software Design-Expert 7.0.0 and Statgraphics Centurion XVI were
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employed. In order to determine the optimum PLE conditions, three independent factors
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were selected using a rotatable CCD: extraction temperature (50-198 °C), extraction
182
time (5-20 min) and water/ethanol ratio (0/100-100/0, v/v) with five levels for each
183
variable. The CCD involved 20 experimental runs: 23 factorial points, 2×3 axial points
184
and 6 center points. The order of experiments was randomized. The runs were carried
185
out in a single block.
186
Another rotatable CCD was also applied to determine the best conditions to
187
maximize rutin concentration in the raffinate attained by SAF. Antisolvent pressure
188
from 15 to 32.1 MPa, and feed flow rate from 0.16 to 0.44 ml/min with five levels for
189
each variable, were used as independent variables. The CCD involved 13 experimental
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runs: 22 factorial points, 2×2 axial points and 5 center points with a randomized
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experiments order.
192
A statistically significant multiple regression relationship between the independent
193
variables and response variables was established. A second order polynomial model
194
(equation 1) of the design was fitted to evaluate extract yield of amaranth leaves, rutin
195
recovery, and rutin concentration in raffinate as a function of independent variables and
196
their interactions.
197
Y=B0+B1X1+B2X2+B3X3+B11X12+B22X22+B33X32+B12X1X2+B23X2X3+B13X1X3+
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Where B0 is the constant, B1, B2, B3 are the linear, B11, B22, B33 are the quadratic, and
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B12, B13, B23 are the interaction coefficients; X1, X2, X3 are the independent variables
200
and is the random error.
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All CCD experiments were carried out by duplicate. Experimental values with more
202
than 5% deviation were replicated three times. Chromatographic measurements of each
203
sample were also determined by duplicate.
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2.5. Ultrasound-assisted extraction (UAE)
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UAE was used as a reference extraction method [28] with slight modifications,
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employing an ultrasonic bath Bandelin (RK 100 H, Berlin, Germany). Two g of milled
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sample was weighed into 100 mL conical flask and mixed with 30 mL (90:10, v/v)
209
methanol/ethanol solution. The flask was sealed, covered from light and placed into the
210
ultrasonic bath for 50 min at room temperature (25 °C). To maintain a constant water
211
temperature, the ultrasonic bath was cooled with ice. Extracts obtained were evaporated
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using rotary vacuum evaporator at 40 °C and placed in the refrigerator until use.
213 214
2.6. HPLC analysis
215
Quantification of rutin was performed by dissolving 0.01 g of crude extracts in 1
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mL of water/methanol mixture (50:50). Analysis conditions were adopted from previous
217
study (7). Before analysis all samples were filtered through a nylon syringe filter
218
(Sartorius, pore size 0.45µm) into HPLC glass vials. An Agilent 1100 series (Agilent
219
Technologies, Waldbronn, Germany) HPLC equipped with an auto sampler and a diode
220
array detector (DAD) was employed. Separation of rutin was accomplished on a C18-
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AR ACE (Advanced Chromatography Technologies, Aberdeen, Scotland) column
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(150×4.6 mm i.d., particle size 5 µm), thermostated at 30 °C in a column oven (Cecil,
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CE 4600). The mobile phases used consisted of 2.5% acetic acid (solvent A) and
224
acetonitrile (solvent B) eluted using the following gradient: 0 min, 10% B; 5 min, 20%
225
B; 10 min, 25% B; 20 min, 45% B; 30 min, 100% B; 35 min, 100% B; 40 min, 10% B,
226
using a flow rate of 0.8 mL/min. The injection volume of was 20 µL.
227
quantification was performed at 350 nm using a calibration curve, which was elaborated
Rutin
10
228
by injecting different concentrations of rutin solutions (0.025 – 0.5 mg/mL) under the
229
same described analytical conditions.
230 231
3. Results and discussion
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3.1. Optimization of PLE of rutin from amaranth
233
As mentioned, RSM was used to maximize extract yield and rutin recovery through
234
the optimization of three independent variables, namely extraction temperature (T),
235
extraction time (t), and water/ethanol ratio in the solvent mixture (r) at five levels. As
236
can be seen in Table 1, the total yield of extract from amaranth leaves varied from 10.2
237
to 49.9 g/100 g dry mater (DM) whereas rutin recovery ranged from 6.95 to 14.03 g/kg
238
DM. Statistically estimated optimum extraction conditions to maximize extraction yield
239
and rutin recovery include the use of 70:30 (v/v) water:ethanol as solvent, at 188°C and
240
20 min as static extraction temperature and time. Observed extraction yield under
241
optimal conditions was 55.8 g/100 g DM (predicted 54.67 g/100 g DM) and rutin
242
recovery was 14.30 g/kg DM (predicted 14.35 g/100 g DM).
243
The model of extraction yield was evaluated by analysis of variance (ANOVA) (see
244
Table 2). The significance of each coefficient was determined using the Student’s t test
245
and p value. The analysis of the quadratic regression model for extract yield from
246
amaranth leaves showed that the model was significant (p˂0.0001). The model showed
247
that the factors with the largest effect on extraction yield were T (p<0.0001) and r
248
(p<0.0001). Interaction effect between T and r was significant (p<0.01) indicating
249
synergistic effect between these factors; other interactions between factors had not
250
significant effects on extract yield (p>0.01). The second-order terms as T2 and r2 were
251
significant (p<0.0001), respectively whereas t2 was not significant (p>0.01).
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252
The determination coefficient (R2=0.996) obtained indicates that the fitted model
253
explains 99.6% of the variability in the extraction yield. The adjusted R-squared value,
254
of 0.993 and predicted R-squared of 0.973 were in good agreement indicating that this
255
design could be used for modeling the response variables employed. The coefficient of
256
variation (CV) of the model was 2.81% meaning that the model can be considered as
257
reproducible.
258
The estimated effects of each factor for extraction yield are summarized in the
259
Pareto chart shown in Figure 3A, arranged in decreasing order of importance. The
260
length of each bar is proportional to the standardized effect, which was calculated
261
dividing the estimated effects by its standard error. Different bar shadings indicate
262
positive and negative effects of the factors in the response variables and the vertical line
263
tests the significance of the effects at the 95 % confidence level. In this case, six effects
264
were significant in the following decreasing order: T, r, r2, t, T2 and Tr. Equation 2 shows the regression model between dependent variable and
265 266
independent variables and their interactions:
267
Y = -1.78-0.06×T-0.62×t+0.51×r+0.00074×T2+0.0021×T×t+0.0008×T×r-0.019×t2-0.0013×t×r-0.0039×r2
268
(2)
269
Where Y is the extraction yield and T, t and r are the coded values of temperature, time
270
and water/ethanol ratio, respectively.
271
Response surface plots showing the effect of extraction time, temperature and
272
water proportion in the solvent mixture on the extraction yield are presented in Figure 4.
273
As can be seen, an increase in the extraction temperature provided higher extraction
274
yields. This fact has been previously reported by other authors [29] since higher
275
extraction temperatures favor faster diffusion rates of analytes and help to reduce the
276
interactions between analytes and sample matrix by disruption of intermolecular forces.
277
It can be observed that the yield is also influenced by water content in the solvent 12
278
mixture; for instance, the extraction yield increased by increasing the water content in
279
the solvent up to 70%. On the other hand, extraction time had a small influence on
280
extraction yield.
281
Statistical analysis of the model of rutin recovery from amaranth leaves was
282
evaluated by ANOVA (Table 2). The model for rutin recovery from amaranth leaves
283
showed that the model was significant (p˂0.0001). The highest effect on rutin recovery
284
was exerted by the solvent composition, r (p<0.0001) followed by time (t) (p<0.01) and
285
temperature (T) (p<0.05). The quadratic effect of water/ethanol ratio showed the most
286
significant influence (p<0.0001) whereas t2 and T2 were not significant (p>0.01). The
287
interaction between T and r was also positive and significant at p≤0.01, indicating a
288
synergistic effect between T and r on rutin recovery. Other interactions between factors
289
did not show significant effects on rutin recovery (p>0.01).
290
The determination coefficient was 0.984, indicating that the fitted model explained
291
98.4% of the variability in rutin recovery whilst the adjusted R-squared (0.969) was in
292
reasonable agreement with predicted R-squared (0.907). The following regression
293
model (equation 3) was obtained in order to predict achievable rutin recovery:
294
Y=6848.15+2.65×T-72.62×t+190.8×r+0.1×T×t+0.25×T×r+0.43×t×r-0.05×T2+4.71×t2-1.88×r2
295
Where Y is the rutin recovery and T, t and r are the coded values of temperature, time
296
and water/ethanol ratio, respectively.
(3)
297
The Pareto chart showed independent variables effects for rutin recovery (Figure
298
3B), arranged in decreasing order of importance. Response surface plots showing the
299
effects of extraction time, temperature and water/ethanol ratio on the solvent mixture on
300
rutin recovery are presented in Figure 5. Figure 5a shows the solvent composition and
301
temperature effects on rutin content. It is evident that an increase in water ratio in the
302
solvent mixture up to 65-70% and an increase on temperature up to 190 °C yielded
13
303
maximum rutin recovery (14.30 g/kg DM); in fact, temperature possessed a minor
304
although significant effect for rutin recovery. Similarly, from Figure 5b it is possible to
305
see that water ratio in the solvent mixture up to 65-70% at the longest tested extraction
306
time yielded the maximum rutin recovery. Figure 5c illustrates the effects of extraction
307
time and temperature. Unlike experiments for optimizing extract yield, solvent
308
composition had the main influence on rutin recovery compared to the effects of
309
extraction temperature and time. Rutin recovery was maximum at 70:30 (water/ethanol)
310
ratio, whereas the lowest rutin content was isolated using a water/ethanol ratio of 0:100.
311
Other extraction parameters such as pressure and time have been previously
312
demonstrated to possess lower effects on extraction rates [29]. These results also
313
suggest that temperature up to 190 °C had no influence on rutin degradation; this can be
314
due to the extraction at high temperature in absence of oxygen, as has been corroborated
315
by Buchner et al. [30] that reported that there was no degradation of rutin in the absence
316
of oxygen on heating at 100 °C in an aqueous solution.
317
Junqing et al. [31] reported that the solubility of rutin in ethanol, depending on
318
solvent temperature, was 380-700 times higher than in water. According to our results
319
rutin recovery increased with increasing water concentration in the mixture with ethanol
320
up to 70%.
321
By analyzing together the two selected response variables, it can be seen that the
322
highest extraction yield and rutin recovery were obtained under the same extraction
323
conditions, which involve the use of 70:30 (v/v) water/ethanol ratio in the solvent
324
mixture, 188 °C as extraction temperature and 20 min of static extraction time. As can
325
be observed in Table 3, at these conditions, total extraction and rutin yields from the
326
amaranth leaves were respectively 10 and 4 times higher than the values obtained using
14
327
conventional UAE. Predicted (14.35 g/kg DM) and experimental rutin recovery values
328
under optimal conditions were not significantly different.
329
Likewise, under these conditions, the use of amaranth seeds as rutin sources was
330
also explored; rutin recoveries from defatted and not defatted seeds of amaranth under
331
optimum extraction conditions were remarkably lower compared to leaves, reaching
332
0.35 and 0.41 g/kg DM, respectively. Using UAE the same trend was observed,
333
obtaining 0.19 and 0.34 mg/kg DM of rutin from defatted and not defatted seeds,
334
respectively.
335
In comparison to our previous experiments [7], the extraction yield from amaranth
336
leaves obtained at optimum PLE conditions was 2.7 times higher for deffated seeds and
337
7.2 times higher for non-deffated seeds than using conventional extraction, whereas
338
rutin recovery was 3.8 and 8.7 times higher, respectively. Results obtained for rutin
339
content were in the range of those previously described for amaranth seeds (0.08 g/kg
340
DW) and leaves (24.5 g/kg DW) [8].
341 342
3.2. Supercritical antisolvent fractionation (SAF) of rutin
343
Once the optimum PLE conditions for the extraction of rutin from amaranth leaves
344
were established, a new procedure based on the use of SAF was studied for the
345
fractionation of this extract to obtain a further enriched fraction. Flavonoids are
346
practically non-soluble in pure supercritical CO2; however their solubility may be
347
increased by adding a co-solvent such as ethanol [32]. SC-CO2 can be used as an anti-
348
solvent to precipitate higher molecular mass polar compounds and as a solvent to
349
extract medium polarity, ethanol-soluble components. Feed mixture (amaranth leaf PLE
350
extract) was separated into two fractions, according to its components’ solubility in SC-
351
CO2 into a concentrated flavonoid fraction as the primary product (raffinate), and an
15
352
ethanol fraction as a secondary product (extract). Previous results obtained by Catchpole
353
et al. [32] demonstrated that fractionation performance depended on the solvent
354
composition during hydro-alcoholic solvent extraction process, the feed solution to
355
supercritical fluid ratio, the solids concentration in the feed and the pressure used for the
356
antisolvent and separator stages. In the present work, the effect of pressure (p) and feed
357
mixture flow rate (f) on rutin recovery was determined by applying a RSM. Table 4
358
shows the results obtained for rutin recovery under the conditions seted by the
359
experimental design.
360
The ANOVA of the experimental design was used for statistical evaluation of rutin
361
recovery in the raffinate. Independent variables as p being the most significant factor
362
influencing rutin recovery (p<0.05) with F value of 19.26 and f was not significant
363
(p>0.05) with F value of 0.73. The model for rutin recovery was significant (p<0.01)
364
with F value of 19.37 by comparing the mean square against an estimate of the
365
experimental error. Mean absolute error was 0.62. The variation coefficient (CV) of the
366
model was calculated as 6.26, supporting high reproducibility of the model. The
367
predicted values of rutin content in raffinate were calculated using the regression model
368
(equation 4). (4)
369
Y=1630.28-200.01×P+33.65×F
370
Where Y is the rutin content in the raffinate and P and F are the coded values of
371
pressure and feed flow ratio respectively. The R-squared (R2=0.9) and the adjusted R-
372
squared (R2=0.8) showed a close agreement between the experimental results and
373
theoretical values.
374
As can be seen, the highest recovery of rutin was obtained by using an intermediate
375
feed mixture flow rate (0.3 mL/min) and low process pressure (15 MPa); rutin
376
concentration was up to 1.5 times higher compared to the native PLE extract. The
16
377
increase of fractionation pressure above 20 MPa resulted in lower rutin recovery in the
378
raffinate. This can be due to the fact that rutin is co-extracted with CO2, resulting in
379
some loss of rutin in the raffinate by increasing pressure at constant temperature. In
380
terms of feed flow rates, the increase of feed flow (up to 0.3 mL/min) resulted in better
381
rutin recovery in the raffinate than using lower flow rates at low pressure. Feed flow
382
rates of 0.4 mL/min resulted in higher rutin recovery whereas feed flow of 0.44 mL/min
383
resulted in lower rutin recovery at lower pressure; this is in agreement with the fact that
384
feed solution amount mixed with CO2 cannot be too high because it decreases the
385
separation performance of medium polarity components that are soluble in the SC-
386
CO2+ethanol. Feed/SC-CO2 ratio above 0.1 resulted in higher rutin recovery at lower
387
pressure.
388
As can be seen in Figure 6, the recovery of rutin in the raffinate varied as a
389
function of feed mixture to CO2 ratio and applied pressure. A linear effect of both
390
variables on the response variable was observed. The 3D surface clearly revealed that
391
pressure had significant effect on rutin recovery. In fact, the concentration of rutin in the
392
raffinate increased by decreasing process pressure.
393 394 395
4. Conclusions A
combined
extraction
process
has
been
developed
coupling
different
396
environmentally friendly advanced extraction techniques. Pressurized liquid extraction
397
using water and ethanol in the solvent mixture was combined with a supercritical
398
antisolvent fractionation step to maximize the final rutin content attainable from
399
amaranth leaves. At optimum PLE conditions (188 °C, 20 min, 70:30 water/ethanol
400
(v/v)) rutin recovery was 4 times higher than using conventional extraction procedures.
401
The optimized SAF protocol, using supercritical CO2 as antisolvent, was able to
17
402
separate the compounds of the PLE extract in two fractions, one of high polarity
403
(dissolved in water, raffinate) and other of medium polarity (dissolved in ethanol,
404
extract). Rutin was recovered in the raffinate with the highest concentration of 22.57
405
g/kg, at 15 MPa using a feed mixture flow rate of 0.3 ml/min. In conclusion, this
406
combined approach has been demonstrated to be useful to recover bioactive components
407
from natural matrices.
408 409
Acknowledgements
410
The financial support for the scientific mission at the Institute of Food Science
411
Research (CIAL-CSIC) was funded by the European Union Structural Funds project
412
“PhD Students Internship to the Overseas Research Centres” within the framework of
413
the Measure for Enhancing Mobility of Scholars and Other Researchers and the
414
Promotion of Student Research (VP1-3.1-ŠMM-01-V) of the Programme of Human
415
Resources Development Action Plan. M.H. thanks MICINN for his “Ramón y Cajal”
416
research contract.
18
417
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antisolvent fractionation of plant extracts, in: A.R.C. Duarte (Ed.), Current Trends
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of Supercritical Fluid Technology in Pharmaceutical, Nutraceutical and Food
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Processing Industries, Bentham Science Publishers, Sharjah, United Arab Emirates,
486
2009, pp. 71-79.
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platyphylla, Separation and Purification Technology 71 (2010) 168-172.
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[26] L. Zheng, P. Qiuhong, C. Xiangyun, D. Changqing, Optimization on anthocyanins
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Biotechnology 19 (2010) 1047-1053.
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[27] N. E. Durling, O. J. Catchpole, S. J. Tallon, J. B. Grey, Measurement and
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modelling of the ternary phase equilibria for high-pressure carbon dioxide-ethanol-
495
water mixtures, Fluid Phase Equilibria 252 (2007) 103-113.
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[28] L. Peng, J. Xiaopin, W. Yuzhi, Z. Hongbin, C. Qingmei, Ultrasonically assisted
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extraction of rutin from Artemisia selengensis Turcz: Comparison with
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conventional extraction techniques, Food Analytical Methods 3 (2010) 261-268.
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[29] M. Herrero, M. Castro-Puyana, J.A. Mendiola, E. Ibañez, Compressed fluids for the
500
extraction of bioactive compounds, Trends in Analytical Chemistry 43 (2013) 67-
501
83.
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[30] N. Buchner, A. Krumbein, S. Rohn, L.W. Kroh, Effect of thermal processing on
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the flavonols rutin and quercetin, Rapid Communications in Mass Spectrometry 20
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(2006) 3229-3235.
505 506 507 508
[31] Z.J. Junqing, B. Peng, W. Yan, Solubilities of rutin in eight solvents at T=283.15, 298.15, 313.15, 323.15, and 333.15 K, Fluid Phase Equilibria 261 (2007) 111-114. [32] O.J. Catchpole, J.B. Grey, K.A. Mitchell, J.S. Lan, Supercritical antisolvent fractionation of propolis tincture, Journal of Supercritical Fluids 29 (2004) 97-106.
509 510
22
511
FIGURE LEGENDS.
512
Fig. 1. Chemical structure of rutin.
513
Fig. 2. Schematic diagram of the supercritical antisolvent fractionation (SAF) system
514
employed.
515
Fig. 3. Standardized Pareto chart showing the most-influencing variables: (A) for
516
extract yield, (B) for rutin recovery.
517
Fig. 4. Response surface plots of PLE showing independent variables effect on extract
518
yield; (a) effect of time and temperature; (b) effect of temperature and water content in
519
the solvent; (c) effect of time and water content in the solvent. The value of the fixed
520
variable has been selected at the mean value of the tested range.
521
Fig. 5. Response surface plots of PLE showing independent variables effect on rutin
522
recovery; (a) effect of temperature and water content; (b) effect of time and water
523
content; (c) effect of time and temperature. The value of the fixed variable has been
524
selected at the mean value of the tested range.
525
Fig. 6. Response surface plot of rutin recovery in the raffinate after the supercritical
526
antisolvent fractionation of the amaranth leaves PLE extract, as a function of applied
527
pressure and feed flow rate.
23
528
Table 1
529
Extraction yield and amount of rutin recovered in the PLE extracts obtained from
530
amaranth at the indicated extraction conditions.
531
Experimental Temperature Time Water / Ethanol ratio runs (°C) (min) (%) 1 124 12 50/50 2 80 17 80/20 3 50 12 50/50 4 124 20 50/50 5 124 12 50/50 6 168 17 20/80 7 124 12 0/100 8 124 12 100/0 9 168 8 20/80 10 80 17 20/80 11 124 12 50/50 12 124 5 50/50 13 80 8 80/20 14 168 8 80/20 15 124 12 50/50 16 80 8 20/80 17 124 12 50/50 18 168 17 80/20 19 198 12 50/50 20 124 12 50/50 Values are represented as a mean ± standard deviation (n=2)
Extraction yield (g/100 g DM) 30.2±0.26 24.4±0.73 19.8±0.57 32.4±0.67 30.0±0.19 30.6±0.39 10.2±0.11 31.3±0.09 27.1±0.30 16.0±0.06 30.7±0.29 26.9±0.77 23.3±0.37 40.5±0.24 30.4±0.11 13.2±0.02 30.2±0.19 44.2±2.58 49.9±0.65 30.0±0.26
Rutin recovery (g/kg DM) 13.46 ±0.53 12.48 ±0.67 12.53 ±0.30 14.03 ±0.10 13.11 ±0.27 10.14 ±1.07 6.95 ±0.01 9.90 ±0.15 9.66 ±0.43 10.50 ±0.20 13.21 ±0.19 12.83 ±0.01 11.85 ±0.49 13.72 ±0.41 13.17 ±0.21 9.78 ±0.45 12.82 ±0.17 12.76 ±0.07 13.59 ±0.30 12.80 ±0.06
532
24
533
Table 2. Analysis of variance for the experimental results from amaranth leaves investigation. Source
Sum of Squares
df
Model (T) Temperature (t) Time (r) Water/ethanol ratio Tt Tr tr T2 t2 r2 Error Total
1737.47 987.36 30.01 481.28 1.36 9.24 0.27 29.63 2.22 181.38 6.37 1744.31
Extraction yield 9 193.05 1 987.36 1 30.01 1 481.28 1 1.36 1 9.24 1 0.26 1 29.62 1 2.22 1 181.38 10 0.637 19 Rutin recovery
Model (T) Temperature (t) Time (r) Water/ethanol ratio Tt Tr tr T2 t2 r2 Error Total
63040000 642200 1746000 18340000 3362 867200 26912 116000 131200 40850000 1029000 64070000
9 1 1 1 1 1 1 1 1 1 10 19
Mean Square
7004000 642200 1746000 18340000 3362 867200 26912 116000 131200 40850000 102900
F-Value
p-Value Prob > F
299.60 1548.44 47.07 754.77 2.13 14.50 0.421 46.47 3.48 284.46
< 0.0001 < 0.0001 < 0.0001 < 0.0001 0.17 0.003 0.532 < 0.0001 0.092 < 0.0001
68.05 6.24 16.96 178.16 0.033 8.43 0.26 1.13 1.27 396.88
< 0.0001 0.031 0.002 < 0.0001 0.860 0.015 0.620 0.313 0.285 < 0.0001
534
25
535
Table 3. Rutin amounts and total extraction yields recovered using PLE under optimum
536
extraction conditions (188 °C, 20 min, 70:30 water/ethanol (v/v)) and UAE (25 °C, 50
537
min, 90:10 methanol/ethanol (v/v)) from amaranth leaves and seeds. Sample
538
Total extraction yield
Rutin amount
(g/100 g DM)
(g/kg DM)
PLE
UAE
PLE
UAE
Leaves
55.8 ± 0.64
5.8 ± 0.07
14.30 ± 0.06
3.62 ± 0.06
Defatted seeds
67.9 ± 1.60
2.8 ± 0.02
0.35 ± 0.05
0.19 ± 0.001
Non-defatted seeds
64.2 ± 0.38
6.5 ± 0.05
0.41 ± 0.003
0.34 ± 0.002
Values are presented as a mean ± standard deviation (n=2)
26
539
Table 4. Rutin amounts determined in the raffinate fraction after supercritical
540
antisolvent fractionation of the amaranth leaves PLE extract. Run
Pressure (MPa)
Feed mixture flow rate, (mL/min)
Feed/SCCO2 ratio (v/v)
Rutin recovery, (g/kg DM)
1 2 3 4 5 6 7 8 9 10 11 12
30 30 25 32.1 20 25 25 25 15 20 25 25
0.4 0.2 0.16 0.3 0.2 0.3 0.3 0.3 0.3 0.4 0.3 0.44
0.122 0.061 0.051 0.091 0.067 0.095 0.095 0.095 0.107 0.134 0.095 0.139
15.25 ± 2.75 14.79 ± 1.70 15.84 ± 0.79 13.80 ± 1.35 16.26 ± 0.78 16.21 ± 1.76 16.30 ± 1.22 16.00 ± 1.47 22.57 ± 2.71 17.70 ± 0.86 16.09 ± 0.99 16.40 ± 1.27
13
25
0.3
0.095
15.90 ± 1.59
Non fractionated hydro-alcoholic mixture (70:30 H2O/EtOH, v/v )
16.00 ± 0.28
541 542
Values are presented as a mean ± standard deviation (n=2)
27
543 544
Figure 1.
28
545 546
Figure 2.
29
547
548 549
Figure 3.
30
550
551 552
Figure 4.
31
553
554 555
Figure 5.
32
556 557
Figure 6.
33
S0896-8446(15)30049-8 http://dx.doi.org/doi:10.1016/j.supflu.2015.06.022 SUPFLU 3370
To appear in:
J. of Supercritical Fluids
Received date: Revised date: Accepted date:
28-3-2015 25-6-2015 25-6-2015
Please cite this article as: Paulius Kraujalis, Petras Rimantas Venskutonis, Elena Ib´an˜ ez, Miguel Herrero, Optimization of rutin isolation from Amaranthus paniculatus leaves by high pressure extraction and fractionation techniques, The Journal of Supercritical Fluids http://dx.doi.org/10.1016/j.supflu.2015.06.022 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
1
Optimization of rutin isolation from Amaranthus paniculatus leaves by high
2
pressure extraction and fractionation techniques
3
Paulius Kraujalisa, Petras Rimantas Venskutonisa, Elena Ibáñezb*, Miguel Herrerob
4 5 6 7 8 9 10 11
a
12
[email protected]
13
TEL: +34 910 017 956
14
FAX: +34 910 017 905
15 16 17
Graphical abstract
18 19
Highlights
20
70/30 (v/v) water/ethanol ratio, at 188°C and 20 min were optimal parameters for
21
Kaunas University of Technology, Department of Food Science and Technology, Radvilėnų pl. 19, LT-50254, Kaunas, LITHUANIA b
Foodomics Laboratory, Institute of Food Science Research (CIAL, CSIC), Nicolás Cabrera 9, Campus UAM Cantoblanco,28049 - Madrid, SPAIN * Corresponding Author (Elena Ibáñez)
rutin recovery.
22
PLE recovered 4 times more of rutin than conventional extraction.
23
Application of SAF increased rutin concentration up to 1.5 times.
24
A PLE-SAF integrated process was successfully optimized and applied.
1
25
Abstract
26
A procedure based on the application of pressurized liquid extraction (PLE) was
27
employed to recover the major Amaranthus spp. flavonoid glycoside rutin from dried
28
ground plant leaves. A central composite design (CCD) was used to optimize rutin
29
extraction; three factors were considered: extraction solvent (water/ethanol ratio, from
30
0/100 to 100/0), extraction time (from 5 to 20 min) and extraction temperature (from 50
31
to 200°C). Optimization of the three extraction parameters revealed that water/ethanol
32
ratio in the solvent mixture was the most-significant factor influencing rutin yield,
33
followed by extraction temperature. The total rutin recovery varied from 6.95 to 14.03
34
g/kg dry matter (DM) depending on the experimental conditions tested. The optimum
35
extraction conditions for the highest rutin content were 70/30 (v/v) water/ethanol ratio,
36
at 188°C and 20 min as static extraction time. In this case rutin recovery from the leaves
37
(14.30 g/kg DM) was 4-times higher compared to that attainable using conventional
38
ultrasound assisted extraction (3.62 g/kg DM) with methanol:ethanol mixture (90:10) as
39
solvent. Rutin recovery from defatted and non-defatted seeds of amaranth under
40
optimum PLE conditions was 35.3 and 41.1 mg/kg DM, respectively, whereas using
41
ultrasound assisted extraction 19.7 and 34.6 mg/kg DM were obtained, respectively.
42
Extracts obtained under optimum conditions were further fractionated by using
43
supercritical antisolvent fractionation (SAF). Extracts of amaranth leaves containing
44
70% water were mixed with supercritical CO2 and separated into two fractions
45
according to the molecular mass and solubility of the contained compounds. A central
46
composite experimental design was applied for the determination of the best
47
fractionation conditions to maximize rutin enrichment. Maximum rutin concentration in
48
raffinate (22.57 g/kg) was recovered at 15 MPa using a feed mixture flow rate of 0.3
49
mL/min.
2
50 51
Keywords: amaranth, rutin, pressurized liquid extraction, optimization,
52
supercritical antisolvent fractionation
53
3
54
1. Introduction
55
Amaranth (Amaranthus spp.) is a multipurpose valuable crop supplying nutritional
56
grains, leafy vegetables for food and animal feed, as well as medicinal herb [1].
57
Amaranth seeds oil has a high content of unsaturated fatty acids, triterpene squalene [2]
58
and
59
Glutein-free amaranth seeds are the source of high quality protein consisting of easily
60
digestible albumins and globulins, which are the main components of highly nutritive
61
amaranth proteins [4]. In comparison with fine wheat flour, whole amaranth flour has
62
high lysine content [5].
natural lipophilic tocopherol antioxidants possessing vitamin E activity [3].
63
Leafy parts of amaranth contain various constituents which possess important effects
64
on processing properties and human health. Green leafy vegetables of amaranth are a
65
rich source of dietary fibers and others functional components [6] such as phenolic
66
acids, flavonoids and their glycosides. In this regard, rutin (quercetin-3-O-rutinoside,
67
Figure 1) is the main glycoside found in all anatomical parts of amaranth, although
68
leaves and flowers contain the highest amount of rutin [7]. The specific content of rutin
69
in amaranth depends on species, variety and growing conditions, among other factors
70
[8].
71
Rutin is a flavonoid found in many plants including buckwheat, kacip fatimah,
72
prayer plant, Flos Sophorae Immaturus, tobacco leaves, etc. [9-13]. Numerous studies
73
have reported different biological and pharmacological properties of rutin either in vivo
74
or in vitro as well as a risk reduction effect on diverse diseases, thus, promoting health
75
[14]. In fact, rutin possesses several biological activities, including cytoprotective,
76
antiplatelet, anti-inflammatory, anti-tumor, and antibacterial activities [15-20]. Due to
77
these positive activities, numerous extraction methods have been developed to obtain
78
rutin from various plant materials. The techniques employed range from conventional
4
79
solid-liquid extraction (SLE) to more sophisticated advanced processes such as
80
supercritical fluid extraction (SFE) [14]. Pressurised liquid extraction (PLE) and
81
subcritical water extraction (SWE) are promising extraction techniques that make use of
82
elevated temperature and pressure to improve the efficiency of the extraction procedure,
83
shortening the extraction time and reducing solvent consumption [21]. On the other
84
hand, supercritical fluids (SCF) are alternative solvents which could be used for
85
advanced separation techniques in different processes. Recently, supercritical
86
antisolvent fractionation (SAF) has been suggested as an interesting alternative to
87
fractionate phenolic compounds from plant extracts taking advantage of the differences
88
in solubility of these compounds in the solvent and in supercritical CO2 [22]. SAF is
89
achieved by the continuous contact between the supercritical carbon dioxide (SC-CO2)
90
and a polar liquid mixture at pressurized conditions [23]. Under these conditions, the
91
SCF is able to dissolve the less polar solvent and the non-polar compounds in the
92
mixture SC-CO2 + solvent, selectively precipitating heavier, more polar compounds that
93
are not soluble in the fractionation medium [24]. Considering the large number of
94
factors involved in the above mentioned techniques, the use of experimental designs to
95
optimize the process conditions is recommended. Response surface methodology
96
(RSM) applied through a central composite experimental design (CCD) is commonly
97
used to evaluate the effects and interactions of multiple factors and is useful for building
98
second order model for response variables [2, 25, 26].
99
Thus, the goal of the present study was to develop an integrated process, firstly by
100
optimizing the PLE conditions (solvent ratio, extraction time and temperature) to
101
maximize the rutin extraction from amaranth leaves, and secondly, by optimizing the
102
SAF conditions of the PLE extract (containing rutin and other plant components such as
103
sugars and proteins) to obtain a fraction highly enriched in rutin. To the best of our
5
104
knowledge, this is the first time when both green techniques (PLE + SAF) are being
105
employed for rutin recovery from amaranth leaves.
106 107
2. Materials and methods
108
2.1. Samples and chemicals.
109
Aerial parts of amaranth (Amaranthus paniculatus) were collected during flowering
110
stage in 2013 in an organic herb farm (Panara village, Varėna district, Lithuania). The
111
plants were dried at 40°C by active ventilation in the dark. Dry leaves of amaranth were
112
ground in a Retsch GM 200 mill (Haan, Germany). Before grinding, stems were
113
separated from leaves and not used for analysis.
114
Extraction solvents used were ethanol, extra pure (Scharlau, Scharlab, Spain),
115
methanol HPLC grade (VWR, Radnor, Pennsylvania, USA), and water purified with a
116
Milli-Q system (Millipore, Bedford, MA, USA). For the chromatographic analysis,
117
solvents used were of HPLC grade. Acetic acid (≥99%) and rutin hydrate (≥94 %,
118
HPLC) were obtained from Sigma Aldrich (Steinheim, Germany).
119 120
2.2. Pressurized liquid extraction (PLE)
121
PLE was performed in an accelerated solvent extractor ASE-200 (Dionex,
122
Sunnyvale, CA, USA) using 2 g of sample, which was mixed with washed sea sand
123
(Panreac, Barcelona) (1:1) and placed in a 11 mL volume stainless-steel cell. Extraction
124
solvents (ethanol and MiliQ water) were sonicated before extraction to remove any
125
possible dissolved air. Extraction cells were equipped with stainless steel frits and
126
cellulose filters at both ends to avoid solid particles in the collection vial. Once the
127
extraction cell was loaded with sample, an instrumentally-preset heating-up step was
128
applied for a given time prior to any extraction. This warming up time changed
6
129
depending on the extraction temperature (i.e., 5 min when the extraction temperature
130
ranged from 50 to 100 °C, and 6, 8 or 9 min if the extraction temperature was 124, 168
131
or 198 °C, respectively). All experimental runs were performed at 10 MPa and used one
132
static cycle. After each extraction, cells were flushed with 60% cell volume of fresh
133
solvent and finally purged 60 s with nitrogen. The extracts were kept at 4 °C until use
134
for analysis. Extraction time, temperature and solvent ratio were studied using a rotable
135
central composite experimental design (see below) covering the entire instrument’s
136
working range.
137 138
2.3. Supercritical antisolvent fractionation (SAF)
139
SAF was performed using a Helix supercritical fluid extractor (Applied
140
Separations, Allentown, PA, USA) with some modifications in order to introduce liquid
141
feed into supercritical carbon dioxide (SC-CO2) flow (Figure 2). The extract obtained
142
by PLE was mixed with SC-CO2 under constant flow and recovered into two fractions.
143
Two pumps were used for the fractionation of liquid mixture: the extract after PLE
144
(liquid feed) was delivered by HPLC pump and CO2 by liquid CO2 pump. Liquid feed
145
was mixed with SC-CO2 stream in the heated pipe just before the precipitation vessel
146
inlet and sprayed into the precipitation vessel through the nozzle to reach vapour-liquid
147
equilibrium. A precipitation vessel was used to separate the two phases in equilibrium;
148
the liquid phase (compounds insoluble in SC-CO2) collected at the bottom of
149
precipitation vessel, whereas SC-CO2 soluble compounds (vapour phase) exited through
150
the top of precipitation vessel and were recovered in the separator vessel. Twenty mL of
151
liquid feed was used for all fractionation experimental runs. The first fraction (raffinate)
152
was recovered in the pressurized stainless steel precipitation vessel, while the second
153
fraction (extract) was collected in a separator at atmospheric pressure. The pressure of
7
154
SC-CO2 and the liquid feed flow rate were determined from a CCD (see below),
155
whereas the temperature of precipitation vessel and separator was constant at 40 °C and
156
30 °C, respectively. CO2 flow rate was kept constant (0.216 kg/h) during all
157
experimental runs. The attained raffinate was freeze-dried whereas the extract was dried
158
under a gentle nitrogen stream. Both fractions were placed in the refrigerator until
159
analysis.
160
Three components system composed by PLE extract (70% water and 30% ethanol)
161
and supercritical CO2 was employed for fractionation of rutin. The ternary system was
162
composed by a supercritical fluid and a polar mixture as well as the compounds that
163
have been fractionated. Optimization of the conditions to achieve the fractionation into
164
the liquid and the gaseous phase (raffinate and extract, respectively) was performed
165
selecting a zone in the ternary phase equilibria (SC-CO2-EtOH-H2O system) in which
166
the mixture could be separated apart. Further details on the mole fraction equilibrium
167
data for this ternary system at 40 ºC and pressures of 100-300 bar can be found
168
elsewhere (27). By analyzing this theoretical data, it can be seen that the ethanol
169
partition coefficients are influenced by the pressure, the mass percentage of water in the
170
feed and the feed/SC-CO2 flow rate ratio. In the present study, pressure and feed/SC-
171
CO2 flow rate (expressed as feed mixture flow rate) have been selected while % of
172
water in the feed was fixed at 70%.
173 174
2.4. Experimental designs
175
Response surface methodology (RSM) was applied twice, firstly to determine the
176
optimum PLE conditions (solvent ratio, extraction time and temperature) to maxime
177
extract yield and rutin recovery, and secondly, to optimize the SAF conditions to
178
fractionate the PLE extract to obtain a new extract highly enriched in rutin. For this
8
179
purpose statistical software Design-Expert 7.0.0 and Statgraphics Centurion XVI were
180
employed. In order to determine the optimum PLE conditions, three independent factors
181
were selected using a rotatable CCD: extraction temperature (50-198 °C), extraction
182
time (5-20 min) and water/ethanol ratio (0/100-100/0, v/v) with five levels for each
183
variable. The CCD involved 20 experimental runs: 23 factorial points, 2×3 axial points
184
and 6 center points. The order of experiments was randomized. The runs were carried
185
out in a single block.
186
Another rotatable CCD was also applied to determine the best conditions to
187
maximize rutin concentration in the raffinate attained by SAF. Antisolvent pressure
188
from 15 to 32.1 MPa, and feed flow rate from 0.16 to 0.44 ml/min with five levels for
189
each variable, were used as independent variables. The CCD involved 13 experimental
190
runs: 22 factorial points, 2×2 axial points and 5 center points with a randomized
191
experiments order.
192
A statistically significant multiple regression relationship between the independent
193
variables and response variables was established. A second order polynomial model
194
(equation 1) of the design was fitted to evaluate extract yield of amaranth leaves, rutin
195
recovery, and rutin concentration in raffinate as a function of independent variables and
196
their interactions.
197
Y=B0+B1X1+B2X2+B3X3+B11X12+B22X22+B33X32+B12X1X2+B23X2X3+B13X1X3+
198
Where B0 is the constant, B1, B2, B3 are the linear, B11, B22, B33 are the quadratic, and
199
B12, B13, B23 are the interaction coefficients; X1, X2, X3 are the independent variables
200
and is the random error.
201
All CCD experiments were carried out by duplicate. Experimental values with more
202
than 5% deviation were replicated three times. Chromatographic measurements of each
203
sample were also determined by duplicate.
9
204 205
2.5. Ultrasound-assisted extraction (UAE)
206
UAE was used as a reference extraction method [28] with slight modifications,
207
employing an ultrasonic bath Bandelin (RK 100 H, Berlin, Germany). Two g of milled
208
sample was weighed into 100 mL conical flask and mixed with 30 mL (90:10, v/v)
209
methanol/ethanol solution. The flask was sealed, covered from light and placed into the
210
ultrasonic bath for 50 min at room temperature (25 °C). To maintain a constant water
211
temperature, the ultrasonic bath was cooled with ice. Extracts obtained were evaporated
212
using rotary vacuum evaporator at 40 °C and placed in the refrigerator until use.
213 214
2.6. HPLC analysis
215
Quantification of rutin was performed by dissolving 0.01 g of crude extracts in 1
216
mL of water/methanol mixture (50:50). Analysis conditions were adopted from previous
217
study (7). Before analysis all samples were filtered through a nylon syringe filter
218
(Sartorius, pore size 0.45µm) into HPLC glass vials. An Agilent 1100 series (Agilent
219
Technologies, Waldbronn, Germany) HPLC equipped with an auto sampler and a diode
220
array detector (DAD) was employed. Separation of rutin was accomplished on a C18-
221
AR ACE (Advanced Chromatography Technologies, Aberdeen, Scotland) column
222
(150×4.6 mm i.d., particle size 5 µm), thermostated at 30 °C in a column oven (Cecil,
223
CE 4600). The mobile phases used consisted of 2.5% acetic acid (solvent A) and
224
acetonitrile (solvent B) eluted using the following gradient: 0 min, 10% B; 5 min, 20%
225
B; 10 min, 25% B; 20 min, 45% B; 30 min, 100% B; 35 min, 100% B; 40 min, 10% B,
226
using a flow rate of 0.8 mL/min. The injection volume of was 20 µL.
227
quantification was performed at 350 nm using a calibration curve, which was elaborated
Rutin
10
228
by injecting different concentrations of rutin solutions (0.025 – 0.5 mg/mL) under the
229
same described analytical conditions.
230 231
3. Results and discussion
232
3.1. Optimization of PLE of rutin from amaranth
233
As mentioned, RSM was used to maximize extract yield and rutin recovery through
234
the optimization of three independent variables, namely extraction temperature (T),
235
extraction time (t), and water/ethanol ratio in the solvent mixture (r) at five levels. As
236
can be seen in Table 1, the total yield of extract from amaranth leaves varied from 10.2
237
to 49.9 g/100 g dry mater (DM) whereas rutin recovery ranged from 6.95 to 14.03 g/kg
238
DM. Statistically estimated optimum extraction conditions to maximize extraction yield
239
and rutin recovery include the use of 70:30 (v/v) water:ethanol as solvent, at 188°C and
240
20 min as static extraction temperature and time. Observed extraction yield under
241
optimal conditions was 55.8 g/100 g DM (predicted 54.67 g/100 g DM) and rutin
242
recovery was 14.30 g/kg DM (predicted 14.35 g/100 g DM).
243
The model of extraction yield was evaluated by analysis of variance (ANOVA) (see
244
Table 2). The significance of each coefficient was determined using the Student’s t test
245
and p value. The analysis of the quadratic regression model for extract yield from
246
amaranth leaves showed that the model was significant (p˂0.0001). The model showed
247
that the factors with the largest effect on extraction yield were T (p<0.0001) and r
248
(p<0.0001). Interaction effect between T and r was significant (p<0.01) indicating
249
synergistic effect between these factors; other interactions between factors had not
250
significant effects on extract yield (p>0.01). The second-order terms as T2 and r2 were
251
significant (p<0.0001), respectively whereas t2 was not significant (p>0.01).
11
252
The determination coefficient (R2=0.996) obtained indicates that the fitted model
253
explains 99.6% of the variability in the extraction yield. The adjusted R-squared value,
254
of 0.993 and predicted R-squared of 0.973 were in good agreement indicating that this
255
design could be used for modeling the response variables employed. The coefficient of
256
variation (CV) of the model was 2.81% meaning that the model can be considered as
257
reproducible.
258
The estimated effects of each factor for extraction yield are summarized in the
259
Pareto chart shown in Figure 3A, arranged in decreasing order of importance. The
260
length of each bar is proportional to the standardized effect, which was calculated
261
dividing the estimated effects by its standard error. Different bar shadings indicate
262
positive and negative effects of the factors in the response variables and the vertical line
263
tests the significance of the effects at the 95 % confidence level. In this case, six effects
264
were significant in the following decreasing order: T, r, r2, t, T2 and Tr. Equation 2 shows the regression model between dependent variable and
265 266
independent variables and their interactions:
267
Y = -1.78-0.06×T-0.62×t+0.51×r+0.00074×T2+0.0021×T×t+0.0008×T×r-0.019×t2-0.0013×t×r-0.0039×r2
268
(2)
269
Where Y is the extraction yield and T, t and r are the coded values of temperature, time
270
and water/ethanol ratio, respectively.
271
Response surface plots showing the effect of extraction time, temperature and
272
water proportion in the solvent mixture on the extraction yield are presented in Figure 4.
273
As can be seen, an increase in the extraction temperature provided higher extraction
274
yields. This fact has been previously reported by other authors [29] since higher
275
extraction temperatures favor faster diffusion rates of analytes and help to reduce the
276
interactions between analytes and sample matrix by disruption of intermolecular forces.
277
It can be observed that the yield is also influenced by water content in the solvent 12
278
mixture; for instance, the extraction yield increased by increasing the water content in
279
the solvent up to 70%. On the other hand, extraction time had a small influence on
280
extraction yield.
281
Statistical analysis of the model of rutin recovery from amaranth leaves was
282
evaluated by ANOVA (Table 2). The model for rutin recovery from amaranth leaves
283
showed that the model was significant (p˂0.0001). The highest effect on rutin recovery
284
was exerted by the solvent composition, r (p<0.0001) followed by time (t) (p<0.01) and
285
temperature (T) (p<0.05). The quadratic effect of water/ethanol ratio showed the most
286
significant influence (p<0.0001) whereas t2 and T2 were not significant (p>0.01). The
287
interaction between T and r was also positive and significant at p≤0.01, indicating a
288
synergistic effect between T and r on rutin recovery. Other interactions between factors
289
did not show significant effects on rutin recovery (p>0.01).
290
The determination coefficient was 0.984, indicating that the fitted model explained
291
98.4% of the variability in rutin recovery whilst the adjusted R-squared (0.969) was in
292
reasonable agreement with predicted R-squared (0.907). The following regression
293
model (equation 3) was obtained in order to predict achievable rutin recovery:
294
Y=6848.15+2.65×T-72.62×t+190.8×r+0.1×T×t+0.25×T×r+0.43×t×r-0.05×T2+4.71×t2-1.88×r2
295
Where Y is the rutin recovery and T, t and r are the coded values of temperature, time
296
and water/ethanol ratio, respectively.
(3)
297
The Pareto chart showed independent variables effects for rutin recovery (Figure
298
3B), arranged in decreasing order of importance. Response surface plots showing the
299
effects of extraction time, temperature and water/ethanol ratio on the solvent mixture on
300
rutin recovery are presented in Figure 5. Figure 5a shows the solvent composition and
301
temperature effects on rutin content. It is evident that an increase in water ratio in the
302
solvent mixture up to 65-70% and an increase on temperature up to 190 °C yielded
13
303
maximum rutin recovery (14.30 g/kg DM); in fact, temperature possessed a minor
304
although significant effect for rutin recovery. Similarly, from Figure 5b it is possible to
305
see that water ratio in the solvent mixture up to 65-70% at the longest tested extraction
306
time yielded the maximum rutin recovery. Figure 5c illustrates the effects of extraction
307
time and temperature. Unlike experiments for optimizing extract yield, solvent
308
composition had the main influence on rutin recovery compared to the effects of
309
extraction temperature and time. Rutin recovery was maximum at 70:30 (water/ethanol)
310
ratio, whereas the lowest rutin content was isolated using a water/ethanol ratio of 0:100.
311
Other extraction parameters such as pressure and time have been previously
312
demonstrated to possess lower effects on extraction rates [29]. These results also
313
suggest that temperature up to 190 °C had no influence on rutin degradation; this can be
314
due to the extraction at high temperature in absence of oxygen, as has been corroborated
315
by Buchner et al. [30] that reported that there was no degradation of rutin in the absence
316
of oxygen on heating at 100 °C in an aqueous solution.
317
Junqing et al. [31] reported that the solubility of rutin in ethanol, depending on
318
solvent temperature, was 380-700 times higher than in water. According to our results
319
rutin recovery increased with increasing water concentration in the mixture with ethanol
320
up to 70%.
321
By analyzing together the two selected response variables, it can be seen that the
322
highest extraction yield and rutin recovery were obtained under the same extraction
323
conditions, which involve the use of 70:30 (v/v) water/ethanol ratio in the solvent
324
mixture, 188 °C as extraction temperature and 20 min of static extraction time. As can
325
be observed in Table 3, at these conditions, total extraction and rutin yields from the
326
amaranth leaves were respectively 10 and 4 times higher than the values obtained using
14
327
conventional UAE. Predicted (14.35 g/kg DM) and experimental rutin recovery values
328
under optimal conditions were not significantly different.
329
Likewise, under these conditions, the use of amaranth seeds as rutin sources was
330
also explored; rutin recoveries from defatted and not defatted seeds of amaranth under
331
optimum extraction conditions were remarkably lower compared to leaves, reaching
332
0.35 and 0.41 g/kg DM, respectively. Using UAE the same trend was observed,
333
obtaining 0.19 and 0.34 mg/kg DM of rutin from defatted and not defatted seeds,
334
respectively.
335
In comparison to our previous experiments [7], the extraction yield from amaranth
336
leaves obtained at optimum PLE conditions was 2.7 times higher for deffated seeds and
337
7.2 times higher for non-deffated seeds than using conventional extraction, whereas
338
rutin recovery was 3.8 and 8.7 times higher, respectively. Results obtained for rutin
339
content were in the range of those previously described for amaranth seeds (0.08 g/kg
340
DW) and leaves (24.5 g/kg DW) [8].
341 342
3.2. Supercritical antisolvent fractionation (SAF) of rutin
343
Once the optimum PLE conditions for the extraction of rutin from amaranth leaves
344
were established, a new procedure based on the use of SAF was studied for the
345
fractionation of this extract to obtain a further enriched fraction. Flavonoids are
346
practically non-soluble in pure supercritical CO2; however their solubility may be
347
increased by adding a co-solvent such as ethanol [32]. SC-CO2 can be used as an anti-
348
solvent to precipitate higher molecular mass polar compounds and as a solvent to
349
extract medium polarity, ethanol-soluble components. Feed mixture (amaranth leaf PLE
350
extract) was separated into two fractions, according to its components’ solubility in SC-
351
CO2 into a concentrated flavonoid fraction as the primary product (raffinate), and an
15
352
ethanol fraction as a secondary product (extract). Previous results obtained by Catchpole
353
et al. [32] demonstrated that fractionation performance depended on the solvent
354
composition during hydro-alcoholic solvent extraction process, the feed solution to
355
supercritical fluid ratio, the solids concentration in the feed and the pressure used for the
356
antisolvent and separator stages. In the present work, the effect of pressure (p) and feed
357
mixture flow rate (f) on rutin recovery was determined by applying a RSM. Table 4
358
shows the results obtained for rutin recovery under the conditions seted by the
359
experimental design.
360
The ANOVA of the experimental design was used for statistical evaluation of rutin
361
recovery in the raffinate. Independent variables as p being the most significant factor
362
influencing rutin recovery (p<0.05) with F value of 19.26 and f was not significant
363
(p>0.05) with F value of 0.73. The model for rutin recovery was significant (p<0.01)
364
with F value of 19.37 by comparing the mean square against an estimate of the
365
experimental error. Mean absolute error was 0.62. The variation coefficient (CV) of the
366
model was calculated as 6.26, supporting high reproducibility of the model. The
367
predicted values of rutin content in raffinate were calculated using the regression model
368
(equation 4). (4)
369
Y=1630.28-200.01×P+33.65×F
370
Where Y is the rutin content in the raffinate and P and F are the coded values of
371
pressure and feed flow ratio respectively. The R-squared (R2=0.9) and the adjusted R-
372
squared (R2=0.8) showed a close agreement between the experimental results and
373
theoretical values.
374
As can be seen, the highest recovery of rutin was obtained by using an intermediate
375
feed mixture flow rate (0.3 mL/min) and low process pressure (15 MPa); rutin
376
concentration was up to 1.5 times higher compared to the native PLE extract. The
16
377
increase of fractionation pressure above 20 MPa resulted in lower rutin recovery in the
378
raffinate. This can be due to the fact that rutin is co-extracted with CO2, resulting in
379
some loss of rutin in the raffinate by increasing pressure at constant temperature. In
380
terms of feed flow rates, the increase of feed flow (up to 0.3 mL/min) resulted in better
381
rutin recovery in the raffinate than using lower flow rates at low pressure. Feed flow
382
rates of 0.4 mL/min resulted in higher rutin recovery whereas feed flow of 0.44 mL/min
383
resulted in lower rutin recovery at lower pressure; this is in agreement with the fact that
384
feed solution amount mixed with CO2 cannot be too high because it decreases the
385
separation performance of medium polarity components that are soluble in the SC-
386
CO2+ethanol. Feed/SC-CO2 ratio above 0.1 resulted in higher rutin recovery at lower
387
pressure.
388
As can be seen in Figure 6, the recovery of rutin in the raffinate varied as a
389
function of feed mixture to CO2 ratio and applied pressure. A linear effect of both
390
variables on the response variable was observed. The 3D surface clearly revealed that
391
pressure had significant effect on rutin recovery. In fact, the concentration of rutin in the
392
raffinate increased by decreasing process pressure.
393 394 395
4. Conclusions A
combined
extraction
process
has
been
developed
coupling
different
396
environmentally friendly advanced extraction techniques. Pressurized liquid extraction
397
using water and ethanol in the solvent mixture was combined with a supercritical
398
antisolvent fractionation step to maximize the final rutin content attainable from
399
amaranth leaves. At optimum PLE conditions (188 °C, 20 min, 70:30 water/ethanol
400
(v/v)) rutin recovery was 4 times higher than using conventional extraction procedures.
401
The optimized SAF protocol, using supercritical CO2 as antisolvent, was able to
17
402
separate the compounds of the PLE extract in two fractions, one of high polarity
403
(dissolved in water, raffinate) and other of medium polarity (dissolved in ethanol,
404
extract). Rutin was recovered in the raffinate with the highest concentration of 22.57
405
g/kg, at 15 MPa using a feed mixture flow rate of 0.3 ml/min. In conclusion, this
406
combined approach has been demonstrated to be useful to recover bioactive components
407
from natural matrices.
408 409
Acknowledgements
410
The financial support for the scientific mission at the Institute of Food Science
411
Research (CIAL-CSIC) was funded by the European Union Structural Funds project
412
“PhD Students Internship to the Overseas Research Centres” within the framework of
413
the Measure for Enhancing Mobility of Scholars and Other Researchers and the
414
Promotion of Student Research (VP1-3.1-ŠMM-01-V) of the Programme of Human
415
Resources Development Action Plan. M.H. thanks MICINN for his “Ramón y Cajal”
416
research contract.
18
417
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antisolvent fractionation of plant extracts, in: A.R.C. Duarte (Ed.), Current Trends
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of Supercritical Fluid Technology in Pharmaceutical, Nutraceutical and Food
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Processing Industries, Bentham Science Publishers, Sharjah, United Arab Emirates,
486
2009, pp. 71-79.
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Optimization of pressurized liquid extraction for spicatoside A in Liriope
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platyphylla, Separation and Purification Technology 71 (2010) 168-172.
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extraction from wine grape skins using orthogonal test design, Food Science and
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Biotechnology 19 (2010) 1047-1053.
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modelling of the ternary phase equilibria for high-pressure carbon dioxide-ethanol-
495
water mixtures, Fluid Phase Equilibria 252 (2007) 103-113.
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extraction of rutin from Artemisia selengensis Turcz: Comparison with
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conventional extraction techniques, Food Analytical Methods 3 (2010) 261-268.
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extraction of bioactive compounds, Trends in Analytical Chemistry 43 (2013) 67-
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the flavonols rutin and quercetin, Rapid Communications in Mass Spectrometry 20
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511
FIGURE LEGENDS.
512
Fig. 1. Chemical structure of rutin.
513
Fig. 2. Schematic diagram of the supercritical antisolvent fractionation (SAF) system
514
employed.
515
Fig. 3. Standardized Pareto chart showing the most-influencing variables: (A) for
516
extract yield, (B) for rutin recovery.
517
Fig. 4. Response surface plots of PLE showing independent variables effect on extract
518
yield; (a) effect of time and temperature; (b) effect of temperature and water content in
519
the solvent; (c) effect of time and water content in the solvent. The value of the fixed
520
variable has been selected at the mean value of the tested range.
521
Fig. 5. Response surface plots of PLE showing independent variables effect on rutin
522
recovery; (a) effect of temperature and water content; (b) effect of time and water
523
content; (c) effect of time and temperature. The value of the fixed variable has been
524
selected at the mean value of the tested range.
525
Fig. 6. Response surface plot of rutin recovery in the raffinate after the supercritical
526
antisolvent fractionation of the amaranth leaves PLE extract, as a function of applied
527
pressure and feed flow rate.
23
528
Table 1
529
Extraction yield and amount of rutin recovered in the PLE extracts obtained from
530
amaranth at the indicated extraction conditions.
531
Experimental Temperature Time Water / Ethanol ratio runs (°C) (min) (%) 1 124 12 50/50 2 80 17 80/20 3 50 12 50/50 4 124 20 50/50 5 124 12 50/50 6 168 17 20/80 7 124 12 0/100 8 124 12 100/0 9 168 8 20/80 10 80 17 20/80 11 124 12 50/50 12 124 5 50/50 13 80 8 80/20 14 168 8 80/20 15 124 12 50/50 16 80 8 20/80 17 124 12 50/50 18 168 17 80/20 19 198 12 50/50 20 124 12 50/50 Values are represented as a mean ± standard deviation (n=2)
Extraction yield (g/100 g DM) 30.2±0.26 24.4±0.73 19.8±0.57 32.4±0.67 30.0±0.19 30.6±0.39 10.2±0.11 31.3±0.09 27.1±0.30 16.0±0.06 30.7±0.29 26.9±0.77 23.3±0.37 40.5±0.24 30.4±0.11 13.2±0.02 30.2±0.19 44.2±2.58 49.9±0.65 30.0±0.26
Rutin recovery (g/kg DM) 13.46 ±0.53 12.48 ±0.67 12.53 ±0.30 14.03 ±0.10 13.11 ±0.27 10.14 ±1.07 6.95 ±0.01 9.90 ±0.15 9.66 ±0.43 10.50 ±0.20 13.21 ±0.19 12.83 ±0.01 11.85 ±0.49 13.72 ±0.41 13.17 ±0.21 9.78 ±0.45 12.82 ±0.17 12.76 ±0.07 13.59 ±0.30 12.80 ±0.06
532
24
533
Table 2. Analysis of variance for the experimental results from amaranth leaves investigation. Source
Sum of Squares
df
Model (T) Temperature (t) Time (r) Water/ethanol ratio Tt Tr tr T2 t2 r2 Error Total
1737.47 987.36 30.01 481.28 1.36 9.24 0.27 29.63 2.22 181.38 6.37 1744.31
Extraction yield 9 193.05 1 987.36 1 30.01 1 481.28 1 1.36 1 9.24 1 0.26 1 29.62 1 2.22 1 181.38 10 0.637 19 Rutin recovery
Model (T) Temperature (t) Time (r) Water/ethanol ratio Tt Tr tr T2 t2 r2 Error Total
63040000 642200 1746000 18340000 3362 867200 26912 116000 131200 40850000 1029000 64070000
9 1 1 1 1 1 1 1 1 1 10 19
Mean Square
7004000 642200 1746000 18340000 3362 867200 26912 116000 131200 40850000 102900
F-Value
p-Value Prob > F
299.60 1548.44 47.07 754.77 2.13 14.50 0.421 46.47 3.48 284.46
< 0.0001 < 0.0001 < 0.0001 < 0.0001 0.17 0.003 0.532 < 0.0001 0.092 < 0.0001
68.05 6.24 16.96 178.16 0.033 8.43 0.26 1.13 1.27 396.88
< 0.0001 0.031 0.002 < 0.0001 0.860 0.015 0.620 0.313 0.285 < 0.0001
534
25
535
Table 3. Rutin amounts and total extraction yields recovered using PLE under optimum
536
extraction conditions (188 °C, 20 min, 70:30 water/ethanol (v/v)) and UAE (25 °C, 50
537
min, 90:10 methanol/ethanol (v/v)) from amaranth leaves and seeds. Sample
538
Total extraction yield
Rutin amount
(g/100 g DM)
(g/kg DM)
PLE
UAE
PLE
UAE
Leaves
55.8 ± 0.64
5.8 ± 0.07
14.30 ± 0.06
3.62 ± 0.06
Defatted seeds
67.9 ± 1.60
2.8 ± 0.02
0.35 ± 0.05
0.19 ± 0.001
Non-defatted seeds
64.2 ± 0.38
6.5 ± 0.05
0.41 ± 0.003
0.34 ± 0.002
Values are presented as a mean ± standard deviation (n=2)
26
539
Table 4. Rutin amounts determined in the raffinate fraction after supercritical
540
antisolvent fractionation of the amaranth leaves PLE extract. Run
Pressure (MPa)
Feed mixture flow rate, (mL/min)
Feed/SCCO2 ratio (v/v)
Rutin recovery, (g/kg DM)
1 2 3 4 5 6 7 8 9 10 11 12
30 30 25 32.1 20 25 25 25 15 20 25 25
0.4 0.2 0.16 0.3 0.2 0.3 0.3 0.3 0.3 0.4 0.3 0.44
0.122 0.061 0.051 0.091 0.067 0.095 0.095 0.095 0.107 0.134 0.095 0.139
15.25 ± 2.75 14.79 ± 1.70 15.84 ± 0.79 13.80 ± 1.35 16.26 ± 0.78 16.21 ± 1.76 16.30 ± 1.22 16.00 ± 1.47 22.57 ± 2.71 17.70 ± 0.86 16.09 ± 0.99 16.40 ± 1.27
13
25
0.3
0.095
15.90 ± 1.59
Non fractionated hydro-alcoholic mixture (70:30 H2O/EtOH, v/v )
16.00 ± 0.28
541 542
Values are presented as a mean ± standard deviation (n=2)
27
543 544
Figure 1.
28
545 546
Figure 2.
29
547
548 549
Figure 3.
30
550
551 552
Figure 4.
31
553
554 555
Figure 5.
32
556 557
Figure 6.
33