Supercritical extraction of neolignans from Piper regnelli var. pallescens

J. of Supercritical Fluids 71 (2012) 64–70

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Supercritical extraction of neolignans from Piper regnelli var. pallescens Caroline Ortega Terra Lemos a , Vitor Augusto dos Santos Garcia a , Renata Menoci Gonc¸alves a , Ivana Correa Ramos Leal b , Vera Lúcia Dias Siqueira a , Lúcio Cardozo Filho a,∗ , Vladimir Ferreira Cabral a a b

Universidade Estadual de Maringá (UEM), Av. Colombo, 5790, bloco D-90, 87020-900, Maringá, Paraná, Brazil Universidade Federal do Rio de Janeiro (UFRJ), Rua Aloísio da Silva Gomes, 50, 27930-560, Macaé, Rio de Janeiro, Brazil

a r t i c l e

i n f o

Article history: Received 29 February 2012 Received in revised form 10 July 2012 Accepted 12 July 2012 Keywords: Piper regnellii Supercritical CO2 extraction Neolignans Antioxidant activity Antibacterial activity Mathematical modeling

a b s t r a c t The aim of this study was to compare the yield and biological potential of Piper regnellii extracts obtained by supercritical fluid extraction (SFE) and by conventional method. The extracts were characterized by the HPLC technique according to the structure type of conocarpan and eupomatenoid-5 neolignans. SFE was performed at temperatures of 313 and 333 K and at pressures ranging from 10.92 to 25.00 MPa. An investigation of antioxidant and antibacterial activities was performed as well. Higher levels of neolignans were found in the extracts obtained by SFE. The results indicate that these extracts showed higher antioxidant activity than those obtained by Soxhlet extraction. All extracts showed low total phenolic contents. The extracts obtained by SFE showed minimal inhibitory concentrations (MIC) ranging from 1.95 to 7.81 ␮g/cm3 against oxacillin-sensitive Staphylococcus aureus (OSSA) and MICs from 0.98 to 7.81 ␮g/cm3 against oxacillin-resistant S. aureus (ORSA). The second-order mathematical model used to describe the extraction kinetics was quite satisfactory. © 2012 Elsevier B.V. All rights reserved.

1. Introduction Piper regnellii var. pallescens is popularly known in Brazil as Pariparoba, Caapeba and Capeba [1,2]. Its roots are used in Brazilian folk medicine to treat liver and spleen obstructions, heal wounds, and reduce swelling and skin irritations [3–5]. The search for chemical constituents from this species has been intensified in recent years due to the fact that these constituents are pharmacologically active. Neolignans present in the root extracts of P. regnellii have been isolated, among which are conocarpan, eupomatenoid-3, eupomatenoid-5 and eupomatenoid-6 [6,7]. The microbiological properties of P. regnellii extracts have been previously described by other authors [1–5]. However, reports concerning the antioxidant activity of these extracts were not found in the literature. Free radicals are responsible for lipid peroxidation in foods and, consequently, their rancid taste. Shibata et al. [8] have suggested that consumption of plant-based foods and drinks could reduce the risk of oxidative damage related to diseases and aging. These health preservation aspects are due to the presence of polyphenols and/or other active substances with antioxidant activity [9]. Another recent research study suggests that these bioactive substances also act to prevent significant diseases including cancer, heart disease and allergies [10]. Moreover, the search for new

∗ Corresponding author. Tel.: +55 44 3011 4749; fax: +55 44 3011 4793. E-mail address: [email protected] (L.C. Filho). 0896-8446/$ – see front matter © 2012 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.supflu.2012.07.003

anti-cancer drugs focuses primarily on natural compounds, as they rarely exhibit severe side effects and efficiently act on a wide range of molecular targets involved in carcinogenesis. As a consequence, there has been increased interest in obtaining bioactive compounds with antioxidant potential, obtained from natural sources [11]. Bioactive compounds belong to different chemical classes and can be obtained by different extraction methods [12,13]. However, there are some points to consider when choosing the method to obtain these molecules, such as the toxicity of the solvents used, stability of the compounds and selectivity of the process [14,15]. Some other authors have already described the advantages of supercritical fluid extraction (SFE) compared to conventional techniques to extract compounds from natural sources [16–20]. The SFE of vegetal bioactive compounds, which have a complex nature, is influenced by process conditions such as temperature, pressure, solvent, solvent flow rate and solubility of the compound of interest. When the solute is in a solid matrix, the extraction kinetics depend on experimental conditions. Temperature and pressure affect the kinetics and control the density and solvation power of the solvent [21]. In this context, this paper aims to study the biological activities and chemical composition of the extracts from the leaves and stems of P. regnellii, obtained by different methods, at low and high pressure. For that purpose, extracts obtained by Soxhlet and SFE were analyzed by high performance liquid chromatography (HPLC) for their conocarpan and eupomatenoid-5 contents. Subsequently, the extracts were subjected to antioxidant analysis based

C.O.T. Lemos et al. / J. of Supercritical Fluids 71 (2012) 64–70 Table 1 Extraction parameters used in the supercritical extraction experiments. Run

Pressure (MPa)

Temperature (K)

CO2 density (kg/m3 )

Flow rate (m3 /min)

1 2 3

10.92 24.41 25.00

313 333 313

681.25 781.12 880.22

3 × 10−6 3 × 10−6 3 × 10−6

on the reduction of the DPPH• (2,2-diphenyl-1-picrylhydrazyl) stable radical, to measure the total phenolic content and investigate antibacterial activity. 2. Materials and methods 2.1. Plant material The leaves and stems of P. regnellii specimens were collected from the “Didactic Garden of Medicinal Plants Professor Irenice Silva” on the campus of the State University of Maringá, in September 2010. An exsiccate was deposited in the herbarium of the State University of Maringá, Brazil, under number HUM 8392. The botanical material was dried in a circulating air oven at a temperature of 313 K. After 72 h of drying, the leaves were separated from the stems and then milled in a knife mill (Marconi, Model MA-580). Tyler sieves (W.S. Tyler, USA) were used to classify the samples according to particle size. The leaves and stems trapped between the 20 and 30-mesh (#20 = 0.850 mm and #30 = 0.600 mm) sieves were chosen for further extractions because yielded great quantity after grinding. Moreover the solid matrix should have an intermediate size because excessive grinding may result in too fine particles that limit the performance of fixed beds (owing to channeling, formation of dead zones, and compaction) [12]. 2.2. Extraction methods 2.2.1. Organic solvent (Soxhlet) The organic solvent extraction was performed by using a Soxhlet apparatus, according to the methodology set by the Adolfo Lutz Institute [22]. The solvents used were dichloromethane (99.6% purity) and hexane (99.6% purity), both from Merck. The yields obtained for each solvent extraction were expressed in relation to the initial dry weight sample. 2.2.2. Supercritical fluid extraction The experiments were performed on a bench scale unit, which basically consists of a CO2 cylinder (Air Liquide, 95% purity), two thermostatic baths, two syringe pumps (ISCO, Model 500D) and one extractor with internal volume of approximately 170 cm3 , as shown in Fig. 1. Additional details about the experimental apparatus and the procedure have been already described by other authors [23–25]. Pressures between 15.00 and 40.00 MPa and temperatures from 313 to 333 K are commonly used for the supercritical extraction of phenolic compounds, terpenoids and other biologically active substances [12]. Therefore, three combinations of these parameters were used for each run, as shown in Table 1. The influence of density was also evaluated, as it is dependent on both temperature and pressure. CO2 density values were obtained by Angus et al. [26]. The extractor was evenly fed with 0.02 kg of leaves or 0.03 kg of stems at the bottom of the extractor, and the remaining extraction cell space was filled with glass beads (inert bed). Thus, the carbon dioxide fed to the extractor originally passed through the inert bed and then through the plant matrix. After reaching the desired temperature for extraction, the pump and extractor were

65

simultaneously pressurized. After operating pressure was reached, the system was left at rest to reach equilibrium (30 min). The extraction was then performed up to 280 and 260 min for leaves and stems, respectively. These times were determined by preliminary tests that through the extraction kinetics showed depletion of soluble components of the mixture, i.e. indicating that the extracted variation in mass/time showed negligible values. Runs were performed in duplicate for all conditions. The yield values presented in this work refer to the average yield. 2.3. Quantification of neolignans The quantification of the neolignans was based on the methodology by Felipe et al. [27], using a high performance liquid chromatography (HPLC) device. The equipment consisted of a Varian 920LC with ultraviolet–visible detector, equipped with a quaternary pump and autosampler, controlled by Galaxie Software. For the calibration curves of the conocarpan and eupomatenoid-5 isolated from P. regnellii leaf extracts, solutions were used at concentrations ranging from10 to 310 ␮g/cm3 in methanol (J.T. Baker, 99.8% purity). The extracts were prepared in methanol at spectroscopic grade (J.T. Baker, 99.8% purity) at 1 mg/mL and filtered through a 0.45 ␮m membrane filter (Millipore, Brazil). A Metasil ODS column was used, 5 mm, 150 mm × 4.6 mm, kept at 303 K. The separation was performed by an isocratic elution system consisting of acetonitrile (J.T. Baker, with 99.99% of purity): 2% aqueous glacial acetic acid (Merck, 100% purity) (60:40, v/v) and a flow rate of 1 cm3 /min. The detection was set at 280 nm and total running time was 20 min. Sample volume injected was 20 ␮L. The analyses were performed in duplicate. 2.4. Antioxidant activity 2.4.1. Method for reducing free radicals (DPPH• ) The DPPH (2,2-diphenyl-1-picrylhydrazyl) spectrophotometric assay was based on the methodology of Brand-Williams et al. [28]. The stable radical DPPH• from Sigma–Aldrich was used as reagent for reduction by antioxidants, changing its color from violet to yellow (proportionally to the concentration of the substance) [29]. The extracts were diluted in methanol to final concentrations ranging from 17 to 250 ␮g/cm3 . An aliquot of 150 ␮L of methanol solution samples at different concentrations was added to 2850 ␮L of DPPH• methanol solution (FMaia, 99.8% purity). To prepare the negative control, 150 ␮L of distilled water (instead of methanol solution sample) was added to the DPPH• methanol solution. Similarly, 150 ␮L of BHT 0.02% (Sigma, analytical standard) (prepared in ethanol solution of FMaia with 95% purity) was used as positive control preparation. The mixtures were left for 1 h in the dark at room temperature and the absorbances were measured at 515 nm in a spectrophotometer (Shimadzu UV-1203) and converted into the percentage antioxidant activity (AA%) as the following formula:

AA % =



1 − Abs. sample Abs. negative control



× 100

A curve was constructed for each extract, where the x-axis represents the final concentration of tested extracts (␮g/mL) and the y-axis expresses the percentage of antioxidant activity. The extract concentration required to reach 50% of AA% (EC50 ) was calculated by linear plot regression. 2.4.2. Total phenolic contents To determine the total phenolic content, the method described by Meda et al. [30] was employed with modifications, using

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Fig. 1. Experimental apparatus used for supercritical CO2 extraction. C, CO2 cylinder; A and B, syringe-type pump; PC1, PC2, pump controller; PG, pressure gauge; TI, temperature indicator; NV, needle valve; MV, micrometric valve; R1, reservoir collection; TB1 and TB2, thermostatic bath.

the Folin–Denis (Sigma–Aldrich, 100% purity) instead of the Folin–Ciocalteau reagent. Solutions of extracts in methanol (FMaia, 99.8% purity) at 0.5 mg/cm3 were prepared. An aliquot of 0.5 cm3 of this solution was added in 2.5 cm3 of solution containing the 10% Folin–Denis in ultrapure water. After 5 min, 2 cm3 of a freshly prepared solution of sodium carbonate 14% were added (Nuclear, 99.99% purity). The mixture was kept in the dark for 2 h, during which time the solution color is expected to change from green to blue. The absorbance was measured at 760 nm in a spectrophotometer (Shimadzu UV-1203), using the same mixture (without extract) as the blank solution. The phenolic concentration was determined by the intersection of the absorbance of the samples with the calibration curve (R2 = 0.9991), which was constructed with standards of pure gallic acid (Vetec) at concentrations from 0.8 to 7 ␮g/cm3 . Total phenolic content was expressed as g of GAE (gallic acid equivalents) per 100 g of extract.

ranging from 0.15 to 300 ␮L/cm3 . The final concentrations of the plant extracts ranged from 0.98 to 500 ␮g/cm3 . MIC was defined as the lowest concentration of extracts or standard antibiotics on which the tested microorganisms did not show visible growth. 2.6. Mathematical modeling of supercritical extraction The kinetic curves using CO2 extraction of neolignans from P. regnellii were modeled using a second-order empirical model that does not require knowledge of the axial concentration profile of the desired chemical species throughout the extraction bed. A previous investigation showed success in modeling the kinetics of supercritical extraction using this type of model [23]. The extracted mass as a function of time was calculated by the equation:

 M=

2.5. Antibacterial activity The minimum inhibitory concentration (MIC) of all plant extracts and reference antibiotics were determined by the microdilution technique in cation-adjusted Mueller–Hinton broth (CAMHB) (DIFCO), according to the recommendations of the Clinical and Laboratory Standards Institute [31]. The evaluation of antibacterial activity of the extracts was performed by using the oxacillin-sensitive Staphylococcus aureus (OSSA-ATCC 29213) and the oxacillin-resistant S. aureus (ORSA-ATCC 43300) reference strains. Oxacillin and vancomycin (Sigma–Aldrich) were used as antibiotics for the quality control test. A culture of each bacterial species grown in Mueller–Hinton broth was diluted in fresh medium to achieve a final concentration of approximately 108 CFU/cm3 (equivalent to 0.5 McFarland). All bacterial cultures were incubated in aerobic conditions. The extracts were prepared at a concentration of 1 mg/cm3 , using 100 ␮L of dimethyl sulfoxide (DMSO Amresco, 99.9% purity) and 900 ␮L of CAMHB as solvents. Both standard antibiotics (vancomycin and oxacillin) were diluted in the plates at concentrations



te

Ceq Qf t,

Cout Qf dt = Ceq Qf t−

0

t < tr Ceq Qf ˛ ˇ

ln(e(zˇ)u + e[−(tu+z)ˇ]/˛u − 1),

(1) t > tr

where L u

(2)

ˇ = kCeq ˛

(3)

 q0 ˛ = bed εCeq

(4)

tr =

In which bed is the bed density (kg/m3 ) calculated as the ratio between the mass of inert material and the bed volume, u is the interstitial velocity (m/min) that is determined using the values of flow rate of CO2 (Qf ) and the bed dimensions, t is the extraction time (min). The bed porosity, ε, is calculated using the following equation: ε=1−

Vsol Vbed

(5)

where Vsol (m3 ) is the volume of solid used in the extractions (leaves or stems) and Vbed (m3 ) is the bed volume. z is the coordinate in the

C.O.T. Lemos et al. / J. of Supercritical Fluids 71 (2012) 64–70

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Table 2 Yields of supercritical and Soxhlet extractions of the leaves and stems of Piper regnellii. Entry

Extraction condition

SFE 1 2 3

10.92 MPa/313 K 24.41 MPa/333 K 25.00 MPa/313 K

SOXHLET 4 5

Hexane Dichloromethane

CO2 density (kg/m3 )

681.25 781.12 880.22

Yield (%)a Leaves

Stems

0.65 ± 0.01A 2.39 ± 0.01B 1.98 ± 0.02B

0.75 ± 0.03A 1.83 ± 0.02B 1.83 ± 0.04B

4.5 ± 0.4C 7.5 ± 0.4D

3.69 ± 0.11E 5.73 ± 0.14F

Different letters indicate statistically significant difference at 5% significance. a Yield (%) = (extracted mass/mass of dry sample) × 100.

axial direction of the bed (m), k is the kinetic constant (m3 /kg min), Ceq is the equilibrium concentration of extract in the solvent (kg extracted/m3 ). Such parameter was obtained from the kinetic curve considering that the initial points (t < tr ) were saturated. q0 (kg/kg inert solid) is the initial quantity of extract concentrated in the solid matrix after the pressurization of the extractor. q0 was determined using the values of yields (extracted mass/mass of dry sample), mass of dry sample (kg), and mass of inert solid (kg). Cout is the concentration of extract in the fluid phase at the extractor exit. The kinetic constant k is the only parameter fitted in the model. For fitting the parameter k, we used a methodology that minimizes a function using a golden section search algorithm in one dimension. For a complete description of this algorithm, see Press et al. [32]. The objective function used minimizes the sum of squared deviations in predicted extracted mass.



n exp

F=

exp 2

(mcalc − mj j

)

(6)

j=1 exp

In this equation mcalc is the calculated extracted mass; mj is j the experimentally obtained mass; n exp is the number of experimental data of the kinetic curve. 3. Results and discussion 3.1. Overall yield of extraction The average values for the yields of all extraction methods evaluated can be found in Table 2. For constant temperature (313 K; entries 1 and 3) an increase in pressure enhances the yield of SFE, for both leaves and stems. For a pressure next to the highest one (entry 2), particularly, an elevation in temperature (333 K) increases the yield of the supercritical extraction for the leaves, although no change was observed for the extraction from stems. An increase in the extraction pressure results in increasing the density and solvating power of the supercritical fluid. In general, the solute vapor pressure increases with temperature but the solvent density decreases. For high pressures, the solvent density changes only slightly with temperature, and as a result the solute vapor pressure will be the main effect. Therefore, the solubility will increase with the temperature for high pressures [12]. This can be observed comparing experiments 2 and 3 in which yield decreases with density for SFE from leaves. Often an increase in pressure can result in a decrease in selectivity as result of the co-extraction of compounds that reduce the purity and give color to the extract. It is also by the possible decrease in selectivity that the higher values yield are presented in the highest pressures. Increasing the density at a constant temperature, the extraction rate and

solubility of the compounds is increased, causing a greater variety of compounds are extracted. The yields obtained by Soxhlet extractions were higher than the yields obtained by SFE, particularly when using dichloromethane as solvent (for both leaves and stems). Benelli et al. [19] attribute this behavior to the higher temperature, solvent recirculation and solute–solvent interactions found in the Soxhlet extraction method. Although soxhlet extraction is widely used in obtaining organic compounds, it has some limitations related to low selectivity and high extraction time. Furthermore, the supercritical extraction is a technique that has high selectivity which may be determined by controlling the density of the solvent, but mainly by the choice of supercritical fluid being used. 3.2. Quantification of conocarpan and eupomatenoid-5 neolignans Table 3 shows the contents of conocarpan and eupomatenoid-5 in the P. regnellii extracts obtained by both supercritical and Soxhlet techniques. It can be seen that pressure exerts considerable influence on the supercritical extraction of conocarpan (entries 6a and 8a), as the highest levels of this substance (in both leaf and stem) were achieved at the highest pressure adopted. However, an increase in temperature (entries 7a and 8a) results in a decrease in the concentration of conocarpan in the extracts from leaves and stems. One can be noticed from Table 3 that eupomatenoid-5 concentrations increased along with increases in both temperature (entries 7b and 8b) and pressure (entries 6b and 8b) increasing. Stem extracts showed higher concentrations of conocarpan. On the other hand, higher concentrations of eupomatenoid-5 were found in the leaf extracts, confirming the data obtained by Felipe et al. [27]. Due the more rigid structure of stem, the diffusion of the solvent beyond the stem cell is generally more difficult, requiring, in some cases, more drastic conditions to extract the same constituent, at the same amount, compared to other tissues. In contrast, leaves are composed by thin tissue layers, generally flexible and easily penetrated, characteristics that are related to their gas diffusion facilities, for example. In the present work, a comparison between the leaves and stems total yields obtained indicated that it was generally higher for leaves (Table 2 – entries 2–5), in both extraction conditions, SFE and Sohxlet. Specifically for eupomatenoid-5, in SFE conditions (Table 3), better yields were obtained from the leaves compared to the stems. In addition, higher pressures facilitated the process, corroborating the explanation above. But in this case, it is important to highlight that, in normal conditions, without pressure, the differences between stem and leaves showed to be not so expressive suggesting the importance of the SFE system. For conocarpan, in normal extraction conditions (without

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Table 3 Concentration of the compounds conocarpan and eupomatenoid-5 in the extracts of Piper regnellii. Entry

Extraction condition

Conocarpan (%)

Eupomatenoid-5 (%)

Leaves

Stems

Leaves

Stems

24.5 ± 0.4c 28.10 ± 0.05f 37.8 ± 0.2g

19.2 ± 0.1䊉 39.3 ± 0.6* 36.1 ± 0.4

15.9 ± 0.5 23.2 ± 0.3 18.2 ± 0.5䊉,♦,

18.7 ± 0.2䊉,♦ 27.5 ± 0.1

17.6 ± 0.5♦, 17.1 ± 0.4

SFE 6 7 8

10.92 MPa/313 K 24.41 MPa/333 K 25.00 MPa/313 K

19.2 ± 0.2a,b 19.9 ± 0.3b 25.3 ± 0.4c

SOXHLET 9 10

Hexane Dichloromethane

8.7 ± 0.2d 17.0 ± 0.3e

18.8 ± 0.4a 30.0 ± 0.1h

Different letters indicate statistically significant difference at 5% significance. Different symbols indicate statistically significant difference at 5% significance.

pressure-Soxhlet), stems already assemble higher contents of this metabolite, suggesting that, even in higher pressure conditions, the proportion tend to maintain the same, in spite of in higher percentages. The results found indicate that supercritical carbon dioxide proved to be the most efficient solvent in terms of neolignan selectivity. 3.3. Antioxidant activity 3.3.1. DPPH• method As can be observed in Fig. 2, an increase in pressure at constant temperature led to higher EC50 values for those extracts obtained by SFE. This result suggests that an increase in pressure probably provided extracts with minor concentrations of the compounds responsible for antioxidant activity. According to Meireles [12] an increased extraction pressure results in increased density and solvating power of the supercritical fluid, as well as in higher interaction between the fluid and the solid matrix. In antioxidant extraction, increased pressure can result in decreased selectivity as a result of the coextraction of compounds that reduce the purity and can confer color. Fig. 2 presents the results of the antioxidant activity in terms of EC50 for all extracts evaluated. The effect of temperature on the antioxidant activity response for extracts obtained by supercritical extraction is distinctive, as it depends on the extract source (stem or leaf). An temperature increase resulted in an increase in EC50 values for stem extracts. The opposite effect was observed for extracts obtained from leaves. Previous satisfactory results obtained for P. regnellii extracts were obtained by Lo et al. [33], who focused on neolignans research. They found that Honokiol and Magnolol neolignans presented

100 90

85.41

80 68.64

EC50 (μg/mL)

70

antioxidant effect up to 1000 times higher than that presented by ␣tocopherol, a standard antioxidant; both compounds also exhibited strong radical scavenging activity. 3.3.2. Total phenolic compounds Table 4 shows the mean values of antioxidant activity expressed in g GAE/100 g of extract. As can be seen in Table 4, the amount of phenolic compounds in the extracts obtained by SFE (entries 11–13) occurred regardless of temperature or pressure variations applied. The low amounts of phenolic compounds in the extracts suggest that there are other substances present in P. regnellii extracts that can be contributing most effectively to the action of scavenging free radicals than the phenolic compounds themselves. The diversity and complexity of the mixtures in plant extracts make it difficult to characterize the compounds and determine their antioxidant activity. Each plant contains various compounds such as vitamins, chlorophyll and phenolic compounds with different antioxidant activities [34]. Further evaluations are needed to verify the contribution of each individual compound in the total antioxidant activity. 3.4. Antibacterial activity The antibacterial activity was considered strong for MIC ≤ 100 ␮g/cm3 . For MICs between 100 and 500 ␮g/cm3 the activity was considered moderate, for MIC between 500 and 1000 ␮g/cm3 the activity was considered weak, and for MIC > 1000 ␮g/cm3 it was considered inactive [4]. Table 5 shows MICs of Piper regnellii extracts obtained by supercritical and conventional extractions. All extracts showed strong antibacterial activity. However, it was observed that the extracts obtained by supercritical extraction (entries 16, 17 and 18) were more active than the extracts obtained by the conventional method (entries 19 and 20). That fact can be explained because the extracts obtained by supercritical technique presented, in general, more content of the compound eupomatenoid-5 (see Table 3), cited by

63.01 58.2

60 50 40

41.23

43.14

55.38 46.86 45.66

40.89

Table 4 Antioxidant activity of extracts from Piper regnellii. Entry

Extraction condition

30 20 10 0

25.00 MPa/313 K Dichloromethane 10.92 MPa/313 K 24.44 MPa/333 K Hexane

Fig. 2. EC50 (␮g/mL) values for the extracts of leaves () and of stems () from Piper regnellii.

Total phenolics (g GAE/100 g of extract) Leaves

Stems

SFE 11 12 13

313 K/10.92 MPa 333 K/24.41 MPa 313 K/25.00 MPa

1.09 ± 0.13a,b 1.2 ± 0.1a,b 1.3 ± 0.1a

1.39 ± 0.14a 1.48 ± 0.18a 1.07 ± 0.15a,b

SOXHLET 14 15

Hexane Dichloromethane

1.03 ± 0.31a,b 0.73 ± 0.14b

1.12 ± 0.12a,b 1.05 ± 0.11a,b

Different letters indicate statistically significant difference at 5% significance.

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Table 5 MICs of Piper regnellii extracts obtained by different methods. Entry

Extraction condition

MIC (␮g/mL) OSSA

ORSA

Leaves SFE 16 17 18 SOXHLET 19 20 21 22

10.92 MPa/313 K 24.41 MPa/333 K 25.00 MPa/313 K Hexane Dichloromethane Oxacilin Vancomycin

Stems

Leaves

Stems

7.81 1.95 3.90

7.81 7.81 7.81

7.81 0.98 7.81

7.81 7.81 7.81

15.62 15.62 – 1.17

15.62 31.25 – –

NT NT 9.37 –

NT NT

NT, not tested; OSSA, oxacilin-sensitive Staphylococcus aureus; ORSA, oxacilin-resistant S. aureus.

Table 6 Experimental conditions and parameters of the model mass transfer proposed for the experimental data. Entry

Parameters

23 24 25 26 27 28 29 30

10.92 MPa/313 K

M (kg) Qf (m3 /min) ε u (m/min) CO2 (kg/m3 ) bed (kg/m3 ) Ceq (kg/m3 ) k (m3 /kg min)

24.41 MPa/333 K Stems

Leaves

Stems

Leaves

Stems

0.0201 3 × 10−6 0.87 0.0054 681.25 117.3 0.221 5.19 × 10−3

0.0301 3 × 10−6 0.84 0.0056 681.25 176.1 0.391 6.27 × 10−3

0.0201 3 × 10−6 0.87 0.0053 781.12 117.6 1.101 8.05 × 10−3

0.03 3 × 10−6 0.84 0.0056 781.12 175.5 1.460 4.19 × 10−3

0.02 3 × 10−6 0.87 0.0054 880.22 117.1 0.998 6.46 × 10−3

0.0301 3 × 10−6 0.84 0.0056 880.22 175.9 1.714 3.21 × 10−3

Pessini et al. [35] as the main responsible for the antibacterial activity in P. regnellii. The values of MICs presented by the extracts obtained by supercritical method are very close to the values reached by Pessini et al. [35] for isolated eupomatenoid-5 (1.56 ␮g/cm3 ) and eupomatenoid-6 (3.12 ␮g/cm3 ) against the same S. aureus strain. Marc¸al et al. [3] evaluated the antibacterial activity of the ethyl acetate extract of this species and obtained a MIC of 16 ␮g/cm3 against ORSA. They also tested eupomatenoid-5 against the same strain and obtained a MIC of 4 ␮g/cm3 . The satisfactory results presented by the extracts obtained under supercritical conditions can be explained by the synergic effect that occurs between the compounds, suggesting that one constituent may potentiate the action of another.

3.5. Mathematical modeling of supercritical extraction The estimated values of kinetic constant k are shown in Table 6 along with other parameter values of the mass transfer model used in this work. It can be seen in Table 6 that, in general, the values of the parameter k (entry 30) in the extraction of neolignans from leaves were higher than those from the extractions. The Ceq values (entry 29) indicate that the extracts obtained from stems presented higher solubility when compared with the extracts obtained from leaves. The kinetics of calculated and experimental SFE of the leaves and stems are also shown in Figs. 3 and 4, respectively, for all experimental conditions.

0.6

0.5

0.5

Extracted mass (g)

0.4

Extracted mass (g)

25.00 MPa/313 K

Leaves

0.3

0.2

0.1

0.4

0.3

0.2

0.1

0.0

0.0 0

50

100

150

200

250

300

Time (min) Fig. 3. Calculated and experimental kinetic curves of extraction from the leaves of Piper regnellii. () 10.92 MPa/313 K, () 24.41 MPa/333 K, () 25.00 MPa/313 K and (–) second-order model.

0

50

100

150

200

250

300

Time (min) Fig. 4. Calculated and experimental kinetic curves of extraction from the stems of Piper regnellii. () 10.92 MPa/313 K, () 24.41 MPa/333 K, () 25.00 MPa/313 K and (–) second-order model.

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As shown in Figs. 3 and 4, the proposed second-order model satisfactorily represents the kinetics of extraction in all investigated conditions. 4. Conclusions This study evaluated the yield and the biological activity of P. regnellii extracts obtained by conventional and supercritical extraction methodologies. The extracts obtained by the supercritical method showed lower yields when compared to the extracts obtained by Soxhlet. However, supercritical extraction proved to be more selective in terms of conocarpan and aupomatenoid-5. Although satisfactory antioxidant activity results were observed by the DPPH• method for the extracts, their total phenolic contents were low. These results suggest that there are other chemical classes contributing more effectively to the antioxidant activity than the phenolic compounds. The antibacterial activity analysis showed that the extracts obtained by supercritical method were more effective against the staphylococcal strains when compared to the extracts obtained at low pressure. The proposed mathematical model proved to be satisfactory in describing the kinetics of supercritical extraction from the leaves and stems of P. regnellii. Acknowledgments The authors are grateful to CAPES (PROCAD-NF, NANOBIOTEC), CNPq and Araucaria Foundation for financial support. References [1] J.M. Barbosa, L.M. Batista, Review of the plants with anti-inflammatory activity studied in Brazil, Brazilian J. Pharmacognosy 15 (2005) 381–391. [2] G.L. Pessini, B.P. Dias Filho, C.V. Nakamura, A.G. Ferreira, D.A.G. Cortez, Neolignanas e análise do óleo essencial das folhas de Piper regnellii (Miq.) C. DC. var. pallescens (C. DC.) Yunck, Brazilian J. Pharmacognosy 15 (2005) 199–204. [3] F.J.B. Marc¸al, D.A.G. Cortez, T.U. Nakamura, C.V. Nakamura, B.P. Dias Filho, Activity of the extracts and neolignans from Piper regnellii against methicillinresistant Staphylococcus aureus (MRSA), Molecules 15 (2010) 2060–2069. [4] F.B. Holetz, G.L. Pessini, N.R. Sanches, D.A.G. Cortez, C.V. Nakamura, B.P. Dias, Screening of some plants used in Brazilian folk medicine for treatment of infectious diseases, Memórias do Instituto Oswaldo Cruz 97 (2002) 1027–1031. [5] P.S. Luize, T.S. Tiuman, L.G. Morello, P.K. Maza, N.T. Ueda, B.P. Dias, D.A.G. Cortez, J.C.P. Mello, C.V. Nakamura, Effects of medicinal plant extracts on growth of Leishmania (L.) amazonensis and Trypanosoma cruzi, Brasilian J. Pharmaceutical Science 41 (2005) 85–94. [6] P.J.C. Benevides, P. Sartorelli, M.J. Kato, Phenilpropanoids and neolignans from Piper regnellii, Phytochemistry 52 (1999) 339–343. [7] A.M. Koroishi, S.R. Foss, D.A.G. Cortez, T.U. Nakamura, C.V. Nakamura, B.P. Dias Filho, In vitro antifungal activity of extracts and neolignans from Piper regnellii against dermatophytes, J. Ethnopharmacology 117 (2008) 270–277. [8] T. Shibata, K. Ishimaru, S. Kawaguchi, H. Yoshikawa, Y. Hama, Antioxidant activities of phlorotannins isolated from Japanese Laminariaceae, J. Applied Phycology 20 (2008) 705–711. [9] B. Aggarwal, S. Shishodia, Molecular targets of dietary agents for prevention and therapy of cancer, Biochemical Pharmacology 71 (2006) 1397–1421. [10] T. Shibata, K. Nagayama, R. Tanaka, K. Yamaguchi, T. Nakamura, Inhibitory effects of brown algal phlorotannins on secretory phospholipase A2s, lipoxygenases and cyclooxygenases, J. Applied Phycology 15 (2003) 61–66. [11] M. Kelkel, M. Schumacher, M. Dicato, M. Diederich, Antioxidant and antiproliferative properties of lycopene, Free Radical Research 45 (2011) 925–940. [12] M.A.A. Meireles, Extracting Bioactive Compounds for Food Products: Theory and Applications, CRC Press, New York, 2009.

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