Supercritical Fluid Extraction of Compounds of Pharmaceutical Interest from Wendita calysina (Burrito)

: Wendita calysina (commonly known as burrito) is an indigenous Paraguayan medical plat, whose essential oil (EO) is characterized by some pharmaceutical properties. In particular, the main component is D-carvone, with anticancer action and antimicrobial properties against various microorganisms. In this work, supercritical CO 2 (SC-CO 2 ) was used for the extraction of volatile compounds from this plant, selecting di ﬀ erent operative conditions to optimize the extract yield and purity. Pressure was varied from 80 to 250 bar, and two CO 2 ﬂow rates (0.8 kg / h and 1.2 kg / h) were tested. The highest EO percentage in the extract was obtained operating at 100 bar and 40 ◦ C, using ground Wendita calysina leaves of 250 µ m. CO 2 ﬂow rate did not inﬂuence the extraction yield, indicating that an internal mass transfer resistance governs this process. The largely prevailing compound identiﬁed in the extract was D-carvone, with a mean percentage up to 90% w / w .


Introduction
The adoption of a correct diet together with regular physical exercise are recognized as useful for the prevention of some human diseases [1][2][3]. For instance, the daily consumption of foods rich in antioxidants can reduce free radical degradation of cells and tissues in an organism [2,4]. Herbal infusions are a possible sources of these bioactive molecules [5,6]; green tea is one of the major examples, widely used particularly in oriental regions due to its medical effects in the prevention and treatment of various diseases, such as cancer, cardiovascular disease, diabetes and obesity [7].
Maceration is the most frequently used traditional technique to produce extracts from a vegetable matter [16]; an organic solvent and, in some cases, water, affine with the compounds of interest, is used at a specific ratio with the starting vegetable matter. This procedure can last from some hours to some days, and the final extract is recovered after solvent evaporation. However, maceration is not a selective extraction technique and the obtained extracts have to be generally post-processed to remove the organic solvent residues and/or impurities [16].
Supercritical fluid extraction (SFE) is a green alternative technique to extract high-value compounds from vegetable matter, avoiding the use of toxic organic solvents that pollute the final product and the environment [17][18][19]. Supercritical fluids and, in particular, supercritical CO 2 (SC-CO 2 ), are characterized by favorable physico-chemical properties (i.e., gas-like diffusivity, liquid-like density, and near to zero surface tension) and reduced cost [20]. Numerous vegetal materials were processed by SFE [21,22]. Parisotto et al. [23] found that the antitumor activity of Cordia verbenacea extracts obtained by SFE was superior in the reduction of tumor cells viability and proliferation with respect to the extract obtained by classical organic solvent extraction with ethanol. Radojković et al. [24] performed SFE process on Morus leaves working at 300 bar and 40 • C. They obtained a linoleic and linolenic acid-rich extract to be used as dietary supplement and food product. Souza et al. [25] determined the effects of temperature and pressure on the extraction yield and chemical composition of extracts from Aloysia gratissima leaves obtained by SFE. Operative temperature was varied from 40 to 60 • C; whereas pressure was from 150 to 200 bar. The chemical analysis showed the presence of guaiol, pinocamphone, caryophyllene oxide and spathulenol as the major chemical components of this plant extract that contributed to a significant reduction of carrageenan-induced paw edema and myeloperoxidase activity. SFE technology can also allow a fractional separation of the extracts, to obtain high purity compounds [19]. This result is possible when process conditions are opportunely selected. Operative pressure and temperature influence SC-CO 2 solvent power through CO 2 density; therefore, depending on the solubility and on the position inside the vegetable matter of the compounds of interest, these parameters should be selected, together with CO 2 flow rate and vegetable matter particle size, with the aim of minimizing the co-extraction of undesired compounds [26]. Another possibility is to perform the fractional extraction, changing the SC-CO 2 solvent power during processing. In particular, depending on the molecular weight (and on the solubility) of the various components, a reduced SC-CO 2 solvent power can be selected in a first step to extract essential oil compounds (low molecular weight compounds); then, in a second step, operative pressure is increased, to extract the other higher molecular weight compounds [26].
Taking into account these considerations, to our knowledge, this work performed SFE of Wendita calysina leaves for the first time, to obtain a high purity essential oil with enhanced healthy properties for pharmaceutical applications. Systematic gas chromatography-mass spectrometry (GC-MS) analyses were carried out on the extracts, to optimize the parameters related to SFE processing and on the starting vegetable matter, to produce a high added-value, solvent free product.

Supercritical Fluid Extraction
Supercritical CO 2 extraction was performed in a lab-scale apparatus equipped with a 400 cm 3 internal volume extractor. CO 2 (purity 99%) was purchased from Morlando Group s.r.l. (Sant'Antimo, NA, Italy). An amount of 100 g of the vegetable matter (mean particle size of 250 µm) was mixed with 85 g of 3-mm glass beads to avoid caking and channeling phenomena in the fixed bed of particles inside the extractor. Extract fractions were recovered using two separation vessels in series, with an internal volume of 200 cm 3 . Cooling of the first separator was achieved using a thermostated bath (Julabo, mod. F38-EH, Seelbach, Germany). The second separator allowed the continuous discharge of the product using a valve located at the bottom of the vessel. A membrane high-pressure pump (Lewa, mod. LDB1 M210S, Mazzo di Rho, Italy) pumped liquid CO 2 at the desired flow rate. CO 2 was heated up to the extraction temperature in a thermostated oven, and its flow rate was monitored by a calibrated rotameter (ASA, mod. N.5-2500, Serval 115022, Sesto San Giovanni, Italy). Temperatures along the extraction apparatus were measured by thermocouples and regulated using PID controllers (mod. 305, Watlow, Corsico (MI), Italy); whereas pressure was measured by test gauges (mod. MP1, OMET, Lecco, Italy). A schematic representation of an SFE plant can be found in [27]. Each experiment was performed in duplicate.

GC-MS Analyses of Compounds from Wendita Calysina Extracts
Wendita calysina extracts obtained by SFE were characterized by gas chromatography-mass spectrometry (GC-MS). The apparatus was a Varian (mod. Saturn 2100T, San Fernando, CA, USA); separation was achieved using a fused-silica capillary column (mod. DB-5, J&W, Folsom, CA, USA) with a 30-m length, 0.25-mm internal diameter and 0.25-µm film thickness. GC conditions were oven temperature at 40 • C for 5 min; then, programmed heating from 40 to 250 • C at 2 • C/min. After 60 min at 250 • C, temperature was increased to 270 • C (speed 2 • C/min) and, finally, from 270 to 290 • C, at 1 • C/min. Helium was used as the carrier gas (1 mL/min). Samples were run in dichloromethane using a dilution factor of 0.0008% w/w. All the components were identified according to the retention time and molecular weight. The same procedure was adopted for the identification of waxes.

Results
The aim of this work was the extraction of a high purity volatile oil from Wendita calysina leaves by SFE. Since this was the first time this technology was applied to Wendita calysina, a preliminary series of experiments was performed using different CO 2 flow rates, to determine the kind of mass transfer resistance governing this process. CO 2 flow rate was varied from 0.8 to 1.2 kg/h; whereas the plant mean particle size was fixed at 250 µm, obtained using the procedure described in Materials and Methods. SFE was performed at 250 bar and 40 • C (ρ CO2 = 0.880 g/cm 3 ); the first separator was set at −14 • C and the second separator was operated at 25 • C and 15 bar. The selection of these values of temperature in the two separators was based on previous works [17,19,28]; the aim was to induce waxes (i.e., paraffins) precipitation in the first separator due to the solubility reduction of these components in SC-CO 2 at low temperature, and the extract recovery in the second separator at conditions near to the ambient ones to avoid thermal stress on the liquid extract.
The extraction yield, measured as the ratio between the weight of the extract collected at regular time intervals and the starting vegetable matter loaded in the extractor, was similar using the two CO 2 flow rates. This information indicates that an internal mass transfer resistance governs this process [20]: i.e., the compounds of interest are located inside the vegetable matter, therefore, the effect of CO 2 flow rate is negligible. Once the kind of extraction mechanism was ascertained, all the following experiments were performed using a CO 2 flow rate of 0.8 kg/h and Wendita calysina leaves particle size of 250 µm.
The second part of the work was focused on the obtainment of a higher essential oil (EO) purity. Operative pressure was varied from 80 to 250 bar and temperature was fixed at 40 • C, to avoid thermal stresses on the vegetable material, and with the aim of determining the effect of SC-CO 2 solvent power. In all experiments, separators' conditions were the same as the preliminary experiments.
SFE was carried out in two steps (fractional extraction): the first one was performed at 80 bar and 40 • C (ρ CO2 = 0.281 g/cm 3 ), the second one at 150 bar and 40 • C (ρ CO2 = 0.780 g/cm 3 ). Extraction was stopped when a negligible increment of the extract was obtained. A final total yield of 0.763% w/w and an EO yield of 0.717% w/w were measured. However, working at 80 bar and 40 • C, due to the reduced SC-CO 2 density, a too low EO extraction yield was obtained (≈0.03% w/w); therefore, these operative conditions were discarded. Moreover, observing the last 100 min of processing, the distance between the total extract yield curve and the EO yield curve tended to enlarge due to the extraction of non-terpenic compounds: i.e., higher molecular weight compounds were co-extracted that do not belong to Wendita calysina essential oil. Waxes extraction yield was 1.91% w/w, measured in the first separator at the end of the supercritical processing.
A further SFE experiment was performed at 250 bar e 40 • C, to determine the effect of a larger SC-CO 2 density on the total and EO yield. However, as expected, increasing SC-CO 2 density and, thus, its solvent power, the final total yield (0.794% w/w) was larger than the one measured working at 150 bar and 40 • C (0.763% w/w); but, also the difference between the final total yield and the EO yield (0.636% w/w) was still larger, due to the co-extraction of other undesired families of compounds (see Figure 1). Therefore, a lower EO purity characterized this extract. In Figure 1, this trend is clearly visible, even if the extraction kinetics were faster (420 min). Waxes collected in the first separator had a yield of 2.02% w/w. SFE was carried out in two steps (fractional extraction): the first one was performed at 80 bar and 40 °C (ρCO2 = 0.281 g/cm 3 ), the second one at 150 bar and 40 °C (ρCO2 = 0.780 g/cm 3 ). Extraction was stopped when a negligible increment of the extract was obtained. A final total yield of 0.763% w/w and an EO yield of 0.717% w/w were measured. However, working at 80 bar and 40 °C, due to the reduced SC-CO2 density, a too low EO extraction yield was obtained (≈0.03% w/w); therefore, these operative conditions were discarded. Moreover, observing the last 100 min of processing, the distance between the total extract yield curve and the EO yield curve tended to enlarge due to the extraction of non-terpenic compounds: i.e., higher molecular weight compounds were co-extracted that do not belong to Wendita calysina essential oil. Waxes extraction yield was 1.91% w/w, measured in the first separator at the end of the supercritical processing.
A further SFE experiment was performed at 250 bar e 40 °C, to determine the effect of a larger SC-CO2 density on the total and EO yield. However, as expected, increasing SC-CO2 density and, thus, its solvent power, the final total yield (0.794% w/w) was larger than the one measured working at 150 bar and 40 °C (0.763% w/w); but, also the difference between the final total yield and the EO yield (0.636% w/w) was still larger, due to the co-extraction of other undesired families of compounds (see Figure 1). Therefore, a lower EO purity characterized this extract. In Figure 1, this trend is clearly visible, even if the extraction kinetics were faster (420 min). Waxes collected in the first separator had a yield of 2.02% w/w. Taking into account these results, another series of experiments was performed at intermediate SC-CO2 solvent power: i.e., 100 bar and 40 °C (ρCO2 = 0.623 g/cm 3 ), to balance a fast extraction and a high EO purity in the extract. Figure 2 reports that a very similar final total yield (0.697% w/w) and EO yield (0.693% w/w) were obtained: i.e., the two curves practically overlap. This result confirms that adopting the opportune SFE operative conditions, it is possible to selectively extract the compounds of interest. Waxes yield was 1.56% w/w in this case. Taking into account these results, another series of experiments was performed at intermediate SC-CO 2 solvent power: i.e., 100 bar and 40 • C (ρ CO2 = 0.623 g/cm 3 ), to balance a fast extraction and a high EO purity in the extract. Figure 2 reports that a very similar final total yield (0.697% w/w) and EO yield (0.693% w/w) were obtained: i.e., the two curves practically overlap. This result confirms that adopting the opportune SFE operative conditions, it is possible to selectively extract the compounds of interest. Waxes yield was 1.56% w/w in this case.
Summarizing these results, SFE confirmed its flexibility in extracting the compounds of interest changing SC-CO 2 solvent power. This peculiarity, coupled with fractional separation, favored the obtainment of a high purity EO in the extract, without the use of toxic organic solvents.
Extracts collected in the first and in the second separator were systematically characterized by GC-MS, as described in Materials and Methods. The extracts composition was in all cases similar and mainly formed by monoterpenes and sesquiterpenes (at the second separator): identification of compounds related to SFE performed at 100 bar and 40 • C is reported in Table 1. In particular, D-carvone, a monoterpene responsible for the characteristic odor of caraway, was the major compound forming the Wendita calysina essential oil, as demonstrated by the GC-MS trace in Figure 3. Its mean amount in the extract represented up to 90% of the EO composition by supercritical processing. Summarizing these results, SFE confirmed its flexibility in extracting the compounds of interest changing SC-CO2 solvent power. This peculiarity, coupled with fractional separation, favored the obtainment of a high purity EO in the extract, without the use of toxic organic solvents.
Extracts collected in the first and in the second separator were systematically characterized by GC-MS, as described in Materials and Methods. The extracts composition was in all cases similar and mainly formed by monoterpenes and sesquiterpenes (at the second separator): identification of compounds related to SFE performed at 100 bar and 40 °C is reported in Table 1. In particular, D-carvone, a monoterpene responsible for the characteristic odor of caraway, was the major compound forming the Wendita calysina essential oil, as demonstrated by the GC-MS trace in Figure  3. Its mean amount in the extract represented up to 90% of the EO composition by supercritical processing.    Waxes collected in the first separator were white, odorless and mainly formed by Nonacosane (C29) 1.227%, Triacontane (C30) 12.616%, traces of Hentriacontane (C31) and Dotriacontane (C32) 86.150%, as shown in Figure 4. These results demonstrate the efficiency of SFE fractional separation Waxes collected in the first separator were white, odorless and mainly formed by Nonacosane (C 29 ) 1.227%, Triacontane (C 30 ) 12.616%, traces of Hentriacontane (C 31 ) and Dotriacontane (C 32 ) 86.150%, as shown in Figure 4. These results demonstrate the efficiency of SFE fractional separation since, in this separator, only waxes (paraffinic compounds) were precipitated; other peaks, related to other compounds were not detected. Moreover, waxes extraction yield increased from 1.56% w/w (@100 bar) to 2.02% w/w (@250 bar) when a larger SC-CO 2 solvent power was tested. Waxes collected in the first separator were white, odorless and mainly formed by Nonacosane (C29) 1.227%, Triacontane (C30) 12.616%, traces of Hentriacontane (C31) and Dotriacontane (C32) 86.150%, as shown in Figure 4. These results demonstrate the efficiency of SFE fractional separation since, in this separator, only waxes (paraffinic compounds) were precipitated; other peaks, related to other compounds were not detected. Moreover, waxes extraction yield increased from 1.56% w/w (@100 bar) to 2.02% w/w (@250 bar) when a larger SC-CO2 solvent power was tested.

Conclusions
Extracts produced by SFE can have a larger added value, since the co-extraction of undesired families of compounds can be minimized. A selective extraction is possible by obtaining a high purity, solvent-free extract. Operating at 100 bar and 40 °C, SFE applied to Wendita calysina leaves was successful in obtaining an extract with a content of D-carvone up to 90% w/w. This result was possible thanks to the addition of a fractional separation scheme that allowed waxes precipitation in the first separator, and pure extract collection in the second one. This product can be used in the pharmaceutical field thanks to its antimicrobial, anticancer, anti-inflammatory, antioxidant and anti-rheumatic properties.
A subsequent study will be focused on the testing of pharmaceutical properties of Wendita calysina leaves extract, in order to determine its efficacy and the doses required.

Conclusions
Extracts produced by SFE can have a larger added value, since the co-extraction of undesired families of compounds can be minimized. A selective extraction is possible by obtaining a high purity, solvent-free extract. Operating at 100 bar and 40 • C, SFE applied to Wendita calysina leaves was successful in obtaining an extract with a content of D-carvone up to 90% w/w. This result was possible thanks to the addition of a fractional separation scheme that allowed waxes precipitation in the first separator, and pure extract collection in the second one. This product can be used in the pharmaceutical field thanks to its antimicrobial, anticancer, anti-inflammatory, antioxidant and anti-rheumatic properties.
A subsequent study will be focused on the testing of pharmaceutical properties of Wendita calysina leaves extract, in order to determine its efficacy and the doses required.