Assessment of composition and biological activity of Arctium lappa leaves extracts obtained with pressurized liquid and supercritical CO2 extraction

Abstract Pressurized liquid extraction (PLE) of Arctium lappa leaves using aqueous ethanol is examined and presented for the first time in this work. In addition, global extraction yields, chemical composition, phenolic compounds and antioxidant activity of the PLE extracts were compared with those obtained by supercritical fluid extraction (SFE). PLE extractions were evaluated at different conditions of pressure (15, 20 and 25) MPa and temperature (313.15, 333.15 and 353.15) K. The maximum global yield (37.40 wt %) was obtained by PLE at 15 MPa and 353.15 K. Furthermore, the PLE extracts contained higher concentrations of chlorogenic acid (1.84%) and rutin (1.46%) and exhibited considerably high DPPH free radical scavenging activity (160.54 μmol Trolox g −1 ). PLE optimum extract exhibited considerable concentrations of phytol, lupeol and amyrin, making this a promising alternative for obtaining biologically active extracts from natural sources such as Arctium lappa leaves.


Introduction
Due to the constant demand of the food and pharmaceutical industries for novel natural sources rich in nutritional and nutraceutical applications, researchers have been looking for the best ways to extract bioactive compounds from plant raw materials. One such example is the medicinal plant Arctium lappa, commonly known as burdock, considered in this study. A. lappa is a plant native to Europe and Asia, and it was rapidly spread to Brazil, where it grows spontaneously in fields, forests and rural areas. Its acclimatization is so natural that this plant is considered an invasive species [1,2].
The different parts of the burdock plant possess antioxidant [3][4][5][6][7], antibacterial [3,[7][8][9] and anti-inflammatory biological activities [7,10,11]. Hence, its study is timely and feasible, moreover taking into consideration the ease and low cost of the plant's cultivation. Still, at present burdock is underused and there are just a few studies published devoted to recovery, characterization and application of its valuable extracts.
Bioactive compounds from Arctium lappa can be obtained by using different extraction methods, e.g. low pressure methods, such as Soxhlet, ultrasound and microwave extraction [6,12,13]. However, the focus at present is on extraction processes that use smaller amounts of solvents, present high extraction yields and have low environmental impact. In view of this, previous studies have examined the potential of a green solvent -supercritical carbon dioxide (scCO 2 ) -to obtain biologically active extracts from A. lappa [3,4], and the addition of ethanol as a co-solvent to change the non-polar nature of the solvent mixture and enhance the extraction of polar compounds [3,4,[14][15][16].
Another extraction technique that has emerged as an alternative to the conventional ones is the pressurized liquid extraction (PLE). The PLE method uses liquid solvents under high pressures and temperatures, normally above the boiling temperature. This favors extraction performance because the solvent remains in the liquid state even at temperatures above its boiling point [17].
High temperatures improve the mass transfer rates, favoring the extraction of solutes from the raw material to the solvent. Furthermore, raising the temperature increases the solubility and diffusivity of the compounds, while the surface tension and viscosity of the solvent are reduced. High pressure, in general, also favors the extraction because the solvent penetration throughout the structure of the solid matrix is facilitated and the solvent density is increased. Thus, the contact of the solvent with the compounds of interest is enhanced [17][18][19].
In this context, the main aim of this work was two-fold: i) to compare the capabilities to obtain extracts from Arctium lappa leaves, in terms of global extraction yield, of two high pressure extraction techniques, PLE and SFE and ii) to compare the quality of the extracts obtained in terms of antioxidant activity, chemical composition and total phenolic content. The results will reveal which of the two techniques performs better in terms of obtaining extracts rich in biologically active compounds.

Sample preparation and raw material characterization
The raw material (Arctium lappa) used in this work was the same used previously [3], The experiments were performed in a bench-scale supercritical extractor unit. The equipment and procedure applied in this study were described in details elsewhere [3,4,[20][21][22]. As shown previously [3], extraction of A. lappa leaves with scCO 2 , using ethanol as a co-solvent, worked well to obtain biologically active extracts. Thus, in this investigation, the scCO 2 with ethanol (scCO 2 + EtOH) was applied again but in a sequential steps extraction. The reason behind that was that in this way an assessment can be made which of the two applications performs better with respect to yield and extracts compositions. Six steps of sequential extractions were carried out with the view to guarantee complete recovery of the extractable material.
The extraction was performed under the optimum operating conditions identified previously, namely 15 MPa and 353.15 K [3], and at those conditions the same sample (the residue of a previous step) was subjected to another extraction with fresh solvents (scCO 2 and ethanol). For all steps the experimental procedure used was similar to that described in detail by Souza et al. [3]. Briefly, about 20 ± 0.2 g of the raw material was mixed beforehand with ethanol at a mass ratio of 2:1 (ethanol mass: mass of solids) and loaded into the extractor; next, the extractor was loaded with CO 2 . The pressure and temperature were adjusted to the set point and the static extraction period was fixed at 60 min, after which the dynamic extraction (extraction period fixed at 25 min) was started using a compressed CO 2 with a flow rate around 2 mL min -1 . At the end of each step there was no ethanol residue in the extraction vessel. Besides that, to further examine the influence of co-solvents on SE efficiency, distilled water was added to ethanol as an auxiliary solvent (hereunder referred as scCO 2 +Aq. EtOH procedure). For this, a hydroalcoholic solution containing water in ethanol at mass ratio of 2:8 (mass fraction of water 24 wt %), was prepared. This extraction was performed in only one step in order to be compared with the first step of the SE without adding water.

Pressurized liquid extraction (PLE)
The experiments were performed in a bench-scale pressurized liquid unit. were used. All pipes and connections were of stainless steel (1/8"). The PLE was performed with aqueous ethanol (water content fixed at 12 wt %) as solvents. About 7.5 g of raw material was used, thus creating a fixed bed inside the extractor (18 mL). The experimental procedure started by adjusting the extraction vessel temperature. After the system reached the temperature required, the needle valve at the exit of the extraction cell was opened so that the solvent, when pumped at a given flow rate (normally 2.0 mL min -1 ), could reach the extraction cell. When the solvent was visualized in the pipe output, the needle valve and the back-pressure regulator were closed thus allowing the system to reach the desired pressure, which was controlled (using the back-pressure regulator) at the set point. At the end of the static extraction time (10 min) equilibrium was reached, thus the needle valve was opened, the pressure was regulated using the back-pressure regulator, and the dynamic extraction started. Preliminary tests with different water content in ethanol were performed (as described in section 3.2, Table 2), which showed that total phenolic compounds and the highest extraction yields were achieved with aqueous ethanol at the mass ratio of 1:9 (mass fraction of water 12 wt %).
Hence, this aqueous ethanol solution was used in all subsequent experiments.  dimethylpolysiloxane). The experimental procedure applied was similar to that described by Souza et al. [3]. Compounds identification was based on the NIST-14 library database, and on comparison with data available in the literature.

Total phenolic content (TPC)
The total phenolic content (TPC) was determined according to the Folin-Ciocalteu method [23], using gallic acid as a standard, as presented previously [3]. The procedure consisted of mixing 0.1 mL of extract (6 mg mL-1), 7.9 mL of distilled water, 0.5 mL of Folin-Ciocalteu reagent and 1.5 mL of 20 % sodium carbonate, in volumetric flasks.
The reaction mixture was kept in the dark for 2 h and then the absorbance was measured at 765 nm. The results were expressed as milligrams of gallic acid equivalent (GAE) per gram of the extract (mg GAE/g of extract).

Antioxidant activity by DPPH assay
DPPH assay was performed, following Souza et al. [3], using the 2,2-diphenyl-1picrylhydrazyl (DPPH) reagent, where the procedure was adapted from Mensor et al. [24]. The results were expressed as the half-maximal inhibitory concentration (IC 50 ), where IC 50 is the concentration (mg mL -1 ) of extract required to inhibit the production of radicals by 50 %, and also given in μmol trolox equivalent g -1 .

Antioxidant activity by FRAP assay
Antioxidant activity was determined according to the procedure described by Barbi et al. [25], according to the method proposed by Benzie and Strain [26]. The results were expressed in μmol trolox equivalent g -1 .

Antioxidant activity by ABTS assay
The method of Re et al. [27] was used to determine the ABTS•+ scavenging activity, according to the procedure described by Barbi et al. [25]. The results were expressed in μmol trolox equivalent g -1 . Results obtained in this study were statistically analyzed using Statistica 7.0®

Supercritical fluid extraction (scCO 2 )
The characteristics of the raw material used in this study were given in a previous work [3]. Dry A. lappa leaves had residual moisture of about 5.97 ± 0.02 wt %, and mean particle diameter, defined by the Tyler series, of (1.3 ± 0.4) × 10 -3 m [3].
For the SEs the same sample was used for all six steps, while in the scCO 2 +Aq. EtOH EtOH procedure are presented in Table 1, where the antioxidant activity and phenolic content results are shown as well. Although the SE achieved a higher yield and extracts with good biological properties, the solvent volume used was very large, approximately five times greater than in the single step extraction. From this point of view, the application of the sequential extraction method might not present great advantages over the single step extraction for this particular raw material.
However, it was found out that for the single step scCO 2 +Aq. EtOH procedure, it was possible to achieve about 9 wt % yield in just 25 min of extraction (Run 7), which was an increase of about 3 wt % as compared to the yield obtained for the same time of extraction using ethanol as a co-solvent. To achieve good efficiency in a short time is very important for energy savings [28]. The same phenomena was observed by Solana et al. [29] and Paes et al. [30]. As expected, the higher yield results for scCO 2 were obtained when water was introduced in the system with the co-solvent. In addition to good extraction yields, the extract obtained contained a high concentration of phenolic compounds (72.32 mg GAE g extract -1 ) when compared to the other ones, probably due to the hydrophilic nature of constituents, such as rutin hydrate [31].
Regarding the extraction using scCO2+EtOH (or Aq. EtOH), it should be noted that, as discussed in [3], in these extractions basically a switch of solvents inside the extractor takes place: starting with CO 2 -expanded ethanol and ending the extraction with pure CO 2 . The kinetic and thermodynamic aspects of this kind of process are very interesting and are a topic of a future research.

Pressurized liquid extraction (PLE)
Among extraction methods, PLE has stood out due to using smaller amounts of solvent [32,33]. In our research, the PLE was used as an alternative technique for obtaining extracts of A. lappa leaves with biological activities. The preliminary tests revealed that the best extraction conditions were at T = 353.15 K and p = 15 MPa, respectively. Table   2 shows the global yield, phenolic content and antioxidant activity of the extracts obtained by PLE performed with different hydroalcoholic concentrations.
Initially, to compare the efficiency of PLE with Soxhlet extraction carried out with water as a solvent, the extractions were carried out with different mass fractions of distilled water in the hydroalcoholic solution (12, 24, 55 wt %). Though it was possible to verify that the addition of water promoted an increase in the extraction yield (Run 1 and 2), still, there was not a significant increase observed for the different hydroalcoholic concentrations (Runs 2-4). In a previous study, Souza et al. [3] showed that the extraction yield of A. lappa leaves is improved in the presence of a polar solvent, indicating that the constituents of this raw material are mostly polar compounds. Therefore, the addition of water to the solvent in PLE resulted in obtaining a maximum extraction yield [34]. TPC values were also improved in the presence of water because, generally, it enhances phenolics extraction [33].
From the results obtained, it can be seen that water addition improved both extraction yield and TPC values, while the antioxidant activities decreased. Therefore, the mass fraction of water in the hydroalcoholic solution was fixed at 12 wt %, and a randomized 2² experimental design was carried out to determine the influence of temperature and pressure.
The different experimental conditions applied to obtain extracts of A. lappa by PLE using the above hydroalcoholic solution are presented in Table 3, where the global yield, phenolic content and antioxidant activity are shown as well. Table 3 demonstrates that all extraction yield values are higher than those obtained by SE (Table 1). It should be noted that in the extractions performed at a fixed temperature (Runs 1-2 and 3-4, respectively) the extraction yield decreased with increasing the pressure, as shown in Figure 3 (a-b). However, at a fixed pressure an increase in the temperature (Runs 1-3 and 2-4, respectively) enhanced the extraction yield, as shown in Figure 3 (c-d). From Figure 3, it can be deduced that within the range of the conditions investigated the effect of the temperature is more pronounced than that of the pressure.

Figure 3
Statistically, the PLE process was influenced significantly (p < 0.05) by both the temperature and pressure variables, as shown in the Pareto chart (Figure 4a), while  3.3. Phenolic compounds identification and antioxidant activity.
As the results of TPC indicated, the A. lappa leaves extracts contain high concentration of phenolic compounds, and of these, analogously to Solana et al. [29], rutin and some phenolic acids were identified in this study. Table 4 shows that the compounds present in higher amounts in all the samples were chlorogenic acid and rutin, while gallic and dihydroxybenzoic acids are in lower concentrations. Sample comparison shows that the higher phenolic concentrations are exhibited by the extracts obtained by the PLE and scCO 2 +Aq. EtOH procedure (Runs 1-5 and Run 9). As expected, the presence of water positively influenced the extraction of phenolic compounds by both techniques (according to the results displayed in Table 1 and Table 2 (Table 3). This fact reinforces the observation made previously that in the PLE process the temperature is the main factor that positively influences the extraction. The same behavior was verified in other studies as well, see for example Pereira et al. [33] for grape marc extracts, Garcia-Mendoza et al. [32] for juçara residues extracts, and Manuel et al. [35] for citrus products extracts. Possibly, this is due to an increase in the solubility and diffusivity at high temperatures of the phenolics in the mixed solvent (ethanol + water), in addition to the decrease in the viscosity and other factors, which might influence the extraction efficiency [17,36].
In order to ascertain whether the extraction process can influence the biological properties of the extracts obtained, the antioxidant capacity was measured as well. The data obtained is presented in Table 4 and Figure 5, where it is clearly demonstrated that the antioxidant results are related with the phenolic compounds, as obtained by Ferrentino et al. [37]. Figure 5 displays the linear correlation between antioxidant capacity (expressed as IC 50 ) and total phenolic compounds (TPC) for the burdock leaves extracts obtained by PLE, which is in complete analogy with the results presented in the previous work using scCO 2

Chemical Composition
In the A. lappa leaves extracts, different compounds were identified by chemical composition analysis -e.g. terpenoids, phenol and esters. Their relative compositions (%) are presented in Table 5.
For the extracts obtained by PLE and SFE with scCO 2 , the compound that has the highest concentration is lupeol (4-36 %). Analogous results were obtained by Souza et al. [3]. Lupeol is a triterpene naturally present in some vegetable matrices and has gained attention because of its high biological effects like anti-inflammatory and anticancer [40][41][42][43][44]. 2,4-di-tert-butyl phenol found in some samples is also of significant importance, because of its beneficial antioxidant and antimicrobial properties [45][46][47].
The high concentrations of phytol and amyrin determined were in complete analogy to the results obtained previously [3]. Phytol is a diterpene that has antioxidant and antiinflammatory properties [48]. Amyrin is a triterpene, which possesses important biological and pharmacological properties [49,50] such as gastroprotective [51], anxiolytic, antidepressant [52], and antinoceptive [53], as well as antioxidant and cytotoxicity activities [54,55], characterizing these extracts with biological activity potential.
In general, it can be confirmed that the extraction techniques and operating conditions applied influenced substantially the extracts composition. For example, the compounds of interest are present in higher concentration in the extracts obtained when scCO 2 was used as a solvent (samples 6-9). Yet, even though the PLE extracts exhibited lower concentrations of the compounds of interest in the overall results, in terms of extraction yield, antioxidant activities and total phenolics the results were quite satisfactory. Hence, it can be concluded that the PLE is a viable approach to obtaining extracts with high biological activities from burdock leaves.

Conclusions
The present work investigates the potential of different methods for extracting