Extraction of essential oil from Cyperus articulatus L. var. articulatus (priprioca) with pressurized CO2

J. of Supercritical Fluids 88 (2014) 134–141

Contents lists available at ScienceDirect

The Journal of Supercritical Fluids journal homepage: www.elsevier.com/locate/supflu

Extraction of essential oil from Cyperus articulatus L. var. articulatus (priprioca) with pressurized CO2 Inaldo Claudio M. da Silva a , Wilson Linhares dos Santos b , Ivana Correa R. Leal c , Maria das Grac¸as B. Zoghbi d , Andresa Carla Feirhmann b , Vladimir Ferreira Cabral b , Emanuel Negrão Macedo a , Lucio Cardozo-Filho b,∗ a

Universidade Federal do Pará(UFPA), Rua Augusto Correa n. 1,Campus do Guamá – Setor profissional, 66075-110-Belém, Pará, Brazil Universidade Estadual de Maringá (UEM), Av. Colombo 5790, bloco D-90, 87020-900 Maringá, Paraná, Brazil c Universidade Federal do Rio de Janeiro, Faculdade de Farmácia, Laboratório de Produtos Naturais e Ensaios Biológicos (Lab. 34), Bloco A, 2◦ andar, 21941-902 Rio de Janeiro, Brazil d Museu Paraense Emílio Goeldi, Belém, Pará, Brazil b

a r t i c l e

i n f o

Article history: Received 16 October 2013 Received in revised form 3 February 2014 Accepted 3 February 2014 Keywords: Antifungal Activity Antibacterial activity Cyperus articulates Mathematical modeling Supercritical extraction

a b s t r a c t The main goal of this study was to assess the yield and the antimicrobial activity of extracts from Cyperus articulatus L. var. articulatus obtained by pressurized carbon dioxide based on their system phase diagram behavior. The extractions were carried out at 313, 323, 333 K temperatures and, 13 and 25 MPa pressures. The extracts were quantified and chemically characterized by using gas chromatography coupled to mass spectrometry technique. The extracts obtained at the following experimental conditions: 333 K and 13 MPa, showed antifungal activity against Cladosporium sphaerospermum ATCC 4464. At 323 K – 25 MPa, and 333 K – 25 MPa, the extracts showed antibacterial activity against Staphylococcus aureus ATCC 25923. To describe the kinetics of extraction with a packed bed, a mathematical model was employed highlighting the transference mechanisms for masses in the pseudo-binary system as follows (1) carbon dioxide and (2) priprioca extract, the monophasic and multiphasic regions. © 2014 Elsevier B.V. All rights reserved.

1. Introduction Cyperus articulatus L.var. articulates is popularly known as “priprioca” in the Amazon Region and Brazil [1]. The underground parts of this species are used for extracting an intensely colored yellow essential oil, with a strong and agreeable fragrance that has economic interest, especially for the perfume and cosmetics industries [2]. In the popular medicine this oil is commonly used for treating hemorrhoids, diarrheas and for body cleanliness [3]. These facts have been increased an interest for studies with priprioca in recent years [1]. Various authors have already described the therapeutic properties of the priprioca extract. Among them, it can be included: antibacterial activity against Staphylococcus aureus and Pseudomonas aeruginosa [4,5], anticonvulsivant [6] and antioxidant activities [7], insecticide against Trilobium confusum and, as an appetite inhibitor for insects [8]. Nevertheless, there is only one

∗ Corresponding author. E-mail addresses: [email protected], [email protected] (L. Cardozo-Filho). http://dx.doi.org/10.1016/j.supflu.2014.02.001 0896-8446/© 2014 Elsevier B.V. All rights reserved.

paper focusing on the phase equilibrium of supercritical fluids by using priprioca extract [9]. Recently, there is a growing interest on the extraction and/or purification of natural products for food, pharmaceutical and cosmetic industries applications throughout the employment of green methodologies. Concerning that, renewed researches has been conducted considering new technological adjustments for products obtainment, that are required in order to attend environmental protection laws. These searches focus on the studies of non-conventional separation techniques in which do not leave solvent residue on the product. Because of this, many research centers have intensified studies on separation processes that employ pressurized fluids as solvents for obtaining residue-free products. Different temperatures and pressures conditions are generally conducted in supercritical technology experiments aiming bioactive products extractions, including essential oils. These are based on heuristic information from close to the critical and supercritical region due to the complex phase behavior in those regions. There are few papers in literature focusing on pressurized fluids or supercritical extraction of bioactive products concerning phase behavior data as preliminary analysis to carry out further optimized extractions.

I.C.M. da Silva et al. / J. of Supercritical Fluids 88 (2014) 134–141

135

Nomenclature FSC C EDO’s CMH ESF CIM CBM L dp ap dL Qfsc t Ed Co Cpo εb εp Ast fsc fsc Dab Dax Kf m E

supercritical fluid mass concentration of oil in the bulk phase (g oil/g CO2 ) differential ordinary equations Müller–Hinton Broth extraction with supercritical fluid minimum inhibitory concentration minimum bactericidal concentration length of packed bed (m) diameter of the solid particle (m) specific surface of the solid (1/m) diameter of packed bed (m) flow of supercritical fluid (m3 /s) extraction time (min) equilibrium constant between the solid and fluid phases initial mass concentration in the bulk phase (g oil/g CO2 ) initial mass concentration in the pore volume (g oil/g CO2 ) empty fraction of the bed empty fraction of the particle area of the section transversal to the solvent flow (m2 /s) density of supercritical fluid (kg/m3 ) viscosity of upercritical fluid (cpoise)) binary diffusion coefficient (m2 /s) axial dispersion coefficient (m2 /s) mass transfer coefficient (m/s) dry solid mass (kg) accumulated extracted mass of oil (g)

In this context, the general objective of this paper was to evaluate the behavior of the extraction kinetic curve of priprioca oil conducted by Supercritical CO2 and, to comprehend the pseudobinary system phase diagram considering (1) carbon dioxide and (2) priprioca extract. We also assessed biological activities, chemical composition, percentage yield and, mass transfer parameters for the pseudo-binary system (1) carbon dioxide and (2) priprioca extract in the monophasic and multiphasic regions.

Fig. 1. Experimental module used in extractions with pressurized fluid: (A) gas cylinder; (B) high pressure pump; (C) extractor; (D) and (E) thermostatic baths; (V1, V2 and V3) needle valves; (V4) micrometric expansion valve; (G) collecting flask; (F) temperature controller on the expansion valve.

ground particles sizes was determined in Tyler standard series strainers with −10/+80 meshes sizes. The average diameter of the particles used in all experiments was 50 meshes. The porous solid matrix density of the solid was determined by a mercury intrusion assay using the equipment Aminco – modelo 5000 psi – USA, obtaining the value of 0.907 g/cm3 . The skeletal density of the solid was determined using a helium pycnometer (Ultrapycnometer 1000, Quantachrome, USA), and the value obtained was 1.36 g/cm3 . From the densities of the porous solid and the skeletal it was possible to determine the porosity of the solids as being 0.333. 2.2. Extraction methods

2.1. Botanical material

2.2.1. Hydro-distillation extraction (HDE) A steam extraction of the underground parts (100 g) of the vegetal species was conducted by using the Clevenger equipment at 373 K during 6 h. The percentage yield of the extract was expressed in relation to the initial dry mass of the sample.

Tubercles and rhizomes of priprioca (C. articulatus var. articulatus) were obtained from an area cultivated for industrial proposes situated in the village of Cruzeirinho – Baixo Acará – Acará Municipality/PA (01◦ 30 79 S latitude and 048◦ 23 36 W de longitude). The vegetal species was identified by a technician from Goeldi Museum and the voucher specimen of the botanical material was deposited in the Herbarium of Goeldi Museum in Belém-Pará-Brasil being registered under the number MG 195586. The tubercles and rhizomes were harvested for approximately eight months after planting in order to be considered ready for extracting the essential oil. The collected material was stored under room temperature for 24 h and, then, packaged in an oven at 303 K (FABBE170-SP/BR) for 12 h in order to dry. The absolute humidity set for the dried material was 52.37%, which is in accordance with the IAL norm, 1985 [10]. The underground portions (tubercles and rhizomes) were ground and classified by the particle size according to the Sauter method [11]. The distribution of the

2.2.2. Supercritical fluid extraction (SFE) The extraction experiments were carried out in a homemade workbench unit (Fig. 1) by using carbon dioxide, CO2 (AIR PRODUCTS, 99.97% purity) in a supercritical state. The experimental unit basically consists of a solvent reservoir, two thermostatic baths, one syringe pumps (Teledyne ISCO 500), a 166.5 mL jacketed extraction vessel, an absolute pressure transducer (Smar, LD301) equipped with a portable programmer (Smar, HT 201) with 0.12 bar precision and a vessel made of glass to place the extract removed from the extraction chamber. Amounts of around 60 g of finely comminuted dry tubercles and rhizomes (52.37% A.H), were placed in the extraction vessel to form a bed of solids supported by two 200 mesh of wire disks at both ends. The solvent was pumped at a constant flow rate of 3 mL/min into the extraction chamber and kept into contact with the bed of comminute tubercles and rhizomes for at least 30 min to allow system stabilization. Sampling was done by interrupting the solvent feed and opening the valves at the bottom

2. Materials and methods

136

I.C.M. da Silva et al. / J. of Supercritical Fluids 88 (2014) 134–141

Table 1 Operational conditions in experiments with CO2 in the supercritical state. Run

Pressure (MPa)

Temperature (K)

Density (g/cm3 )

Flow (mL/min)

Phase type

C1 C2 C3 C4

25 25 25 13

313 323 333 333

0.880 0.835 0.788 0.507

3 3 3 3

Mono Mono Mono Two

of the chamber (see Fig. 1) from where the mass of extracted oil was removed. The entire procedure was carried out for 2 h by taking samples of extracted oil in 10 min time steps. Solvent density at the current operating conditions was calculated according to Angus et al. [12]. The analysis of variance was performed using the SAS 9.1 software (SAS Institute). Table 1 presents the operating conditions employed in the experiments with carbon dioxide in the supercritical state. The values were established based on the knowledge about the phase’s behavior in the pseudo-binary system (1) CO2 + (2) priprioca oil previously determined by Moura et al. [9]. Extractions were performed in monophasic and multiphasic regions to assess the yield, the chemical composition of the extracts and the mechanisms for transferring masses from extractions. All extractions were performed in duplicate.

The differential equations of mass balances as well as the contour conditions and initials employed in modeling the kinetics of supercritical extraction are represented by Eqs. (1)–(4). Fluid phase:

(1) Solid phase

  ∂Cp ap  kf Cp − C = − ∂t εp + (1 − εp )Ed Subject to the following initial and contour conditions: t = 0 ⇒ C = C0 ;

2.3. Chemical composition by gas chromatography (GC) The qualitative analyses were performed in a GC–MS Shimadzu QP-2010 Plus interface and electron impact, with Rtx-5MS (L = 30 m; d = 0.25 mm) capillary column, using helium as carrier gas with a flow rate of 1.2 mL/min. The oven temperature was programmed to range from 333 to 513 K (3 K/min); the source temperature was set at 523 K and the injector temperature and connections at 523 K. The injection was programmed for no flow division (1 ␮L of a solution at 1.0 mg/1 mL hexane). The quantitative analyses were performed in a GC–FID Shimadzu QP 2010, operating at the same conditions as the GC–MS equipment and using hydrogen as carrier gas. 2.4. Mathematical modeling for the SFE process The extraction process using supercritical fluid in a packed bed was mathematically modeled for the two phases considered following hypotheses: (i) The oil contained in the solid matrix was considered as being in equilibrium with that in its internal pores; thus there is a relation of equilibrium between the concentrations; (ii). The oil contained in the pores of the particle migrates by diffusion to the supercritical fluid (SCF) outside the particle; (iii) The concentrate moves in the direction of the SCF flow, enriching the oil received in the pore phase by diffusion; (iv) There is also a mass diffusion phenomenon countering displacement of SCF in the region around the particle, since it is more concentrated as it runs along the packed bed; (v) Through this process, the oil continuously abandons the solid matrix and transfers to the SCF. The mathematical model employed is based on integrating the differential mass model for the fluid (1) and solid (2) phases, with the initial and surrounding conditions (3) and (4). To solve the mathematical model we applied the Generalized Integral Transform Technique (GITT). The GITT [13–15] has been shown to be a promising tool for solving a great variety of engineering problems that involve convection–diffusion. This technique is based upon the use of expansions in terms of orthogonal auto-functions to express unknown dependent variables. However, unlike the Classical Integral Transform Technique, transformation of the original problem does not need to lead to a decoupled system, which makes the method largely applied [16].

(2)

CP = CP0

Cp = Cp0 to t = 0

(3a) (3b)

Subject to the following initial condition (4a)

(4b)

In which, εp = porosity of the particles, Ed = equilibrium coefficient between the phases, C = mass concentration of solute in the bulk phase, Vs = constant velocity of the solvent (m/s), = axial distance (m), Dax = axial dispersion coefficient (m2 /s), t = time, εb = empty fraction of the bed, ap = surface area of the particle contained in the volume unit (1/m), kf = mass transfer coefficient (m/s), Cp = mass concentration of solute in the volume of the pore, L = total height of the bed (m), Cpo = initial mass concentration in the pore volume, Co = initial mass concentration in the bulk phase. The process of supercritical extraction using an inert solid matrix involves a porous packed bed medium to ensure conditions of predetermined constant temperature and pressure. As demonstrated in Eq. (1), the term ∂C represents the variation of mass with ∂t time; the term

represents the rate of transport of the mass

by convection in the bulk phase. On the right side, the term represents the rate of solute transported by diffusion and the term (1−εb ) εb ap Kf (Cp − C) is the rate of transport of the pore volume to the bulk phase. Defining the following dimensionless groups: =

C − Co Cpo

p =

Cp − Cpo Cpo

(5)

(6) (7)

I.C.M. da Silva et al. / J. of Supercritical Fluids 88 (2014) 134–141

=

Vs t L

(8)

Pe =

Vs L Dax

Sh =

Kf dp Dab

(10)

wo =

Co Cpo

(11)

(9)

ap Kf L Vs



(12) (13)



= εp + 1 − εp Ed

(14)

These were applied in Eqs. (1) and (2) of the fluid and solid phases, respectively, in order to give rise to the following system of partial differential equations:

 ∂ ∂ 1 ∂2  (1 − εb )  + + ˛ p −  + ϕ = 2 Pe ∂Z εb ∂ ∂Z

(15a)

 ∂p ˛ p −  + ϕ =− ∂

(15b)

 = 0 →  = p = 0

(15c)

Z=0→− Z=1→

spore suspension by filtration through sterile gauze. Next, dilutions were made by using dextrose Saboraud broth in order to obtain a final concentration of 1–2 × 106 spores/mL which was estimated using a hemocitometer (Newbauer chamber). This solution (5 ␮L) was carried out to microplates adopting the same dilution procedure as described above. The plates were performed in triplicate and then incubated at 310 K, by 5 days. 3. Results and discussion

ϕ = 1 − wo ˛=

137

∂ + Pe  = 0 ∂Z

∂ = 0, ∂Z



(15d)



∂p =0 ∂Z

(15e)

A problem is understood as a direct problem (well-stated problems) when its initial and contour conditions as well as all other parameters that appear in the formulation are known. The direct problem employed here was solved by applying GITT. 2.5. Antifungal and antibacterial tests The antimicrobial susceptibility tests with priprioca extracts obtained by supercritical fluid were performed by using the broth micro dilution method [17,18]. For the antifungal susceptibility test it was applied a reference method (M27-A3/NCCLS) [17] developed through an inter-laboratory process. The M100-S22/NCCLS [18] method used for the antimicrobial analysis includes the M02-A11 (Performance norms for tests of antimicrobial susceptibility) and M07-A9 (Method for antimicrobial sensitivity tests for aerobically growing bacteria) protocols. The bacteria sample (S. aureus ATCC 25923) was initially growth in a Müller–Hinton broth (MB) medium [DIFCO® ] for 24 h. Then, the culture was diluted in a tube containing sterile saline solution (NaCl – 0.9%) until formation of turbidity identical to the 0.5 McFarland scale (108 Colony Formation Unit (UFC)/mL). The solution was diluted (1:10) in a sterile eppendorf tube containing MB obtaining 107 UFC/mL suspension. From this, 5 ␮L solution were carried out to the microplates assembling 100 ␮L of the extract previously solubilized in DMSO and, 100 ␮L of MB, achieving final concentrations of the extracts that varied from 1000 to 15.625 ␮g/mL. The microplates were performed in triplicate and then incubated at 310 K, by 24 h, in order determine the minimum inhibitory concentration (MIC) of the extracts responsible for the bacteria growth inhibition. The fungal spore suspension (Cladosporium sphaerospermum ATCC 4464) was prepared by washing the fungus culture (previously grown in inclined malt agar for one week) with 4 mL of sterile distilled water. The mycelium fragments were removed from the

3.1. Chemical composition of priprioca extract obtained by SFE and HDE In the priprioca extract obtained by SFE there were identified the following secondary metabolite classes by GC–MS: oxygenated monoterpenes and hydrocarbonated/oxygenated sesquiterpenes. By HDE there were detected the same chemical classes with the addition of hydrocarbonated monoterpenes. Table 2 shows the chemical composition of priprioca extracts (SFE) and the essential oil (HDE). The oxygenated sesquiterpenes found in both experimental conditions presented the greatest relative area percentage (39–70%) compared to the other substances detected in the priprioca extract. Differently from the profile described in literature we did not detect the presence of ␣-pinene and ␤-pinene in the oil samples obtained by SFE. The terpenes mustacone, ␣-ciperone, corimbolone and cariofilene oxide were the constituents identified in this study, as well as in the work described by Moura et al. [9] and/or Zoghbi et al. [1]. A high degree of mustacone was identified in the present work in SFE samples as well as in previous work performed by Zoghbi [1] in HDE samples. Among the substances mentioned above, ␣-ciperone and cariofilene oxide were identified in all papers so far presented in the literature for priprioca. The high level of cariofilene oxide (together with ␣pinene and mustacone) was reported as being characteristic of the essential oil of C. articulatus cultivated in the State of Pará/Brazil [19] 3.2. Kinetics and overall yield for the extractions The supercritical fluid extraction (SFE) conditions were conducted based on data for the phase transition (Fig. 2) of the CO2 pseudo-binary system (1) priprioca extract (2) determined by Moura et al. [9]. It is recognized that the knowledge of the system phases assembling carbon dioxide and essential oils is an important parameter for defining the temperature and pressure operational conditions in SFE. However, there are few studies directly using that information for conducting extraction experiments using pressurized fluids. Generally, the conditions for extracting essential oils employing pressurized fluids are defined based on prior experiments or by the experience of the research group itself which limit the technique in some aspects (yield low and energy cost high). Based on the phase diagram, the points of oil solubilization are found in the region rich in CO2 . Compared to a region with two or three phases in contact in the extraction phase, a better oil yield is expected in the monophasic region because of a facilitated mass transfer. For the system being studied, as can be seen in Fig. 2, the experimental conditions were conducted in a single phase and in a multiphasic region. The end time for each experiment with pressurized fluid was defined based on the condition in which the oil mass extracted did not present a significant variation according to the time. In order to compare the values of the yields obtained in each experiment a fixed time of 110 min was chosen. The solvent flow for supercritical CO2 was set at 3 mL/min. In Fig. 3 shows kinetics

138

I.C.M. da Silva et al. / J. of Supercritical Fluids 88 (2014) 134–141

Table 2 Chemical composition of priprioca extracts (SFE) and essential oil (HDE). Compound

Retention time (min)

Area % C1

␣-Pinene tuja 2.4(10)-Diene ␤-Pinene para-Cimene para-Cimenene trans-Pinocarveol Pinocarvone Terpinen-4-ol para-Cimen-8-ol ␣-Terpineol Mirtenol Verbenone trans-Carveol ␣-Copaene Ciperene Rotundene Eudesma-2.4.11-trieno ␤-Selinene ␣-Bulnesene trans-Calamenene Cariofilene oxide Humelene epoxide II Patchulenone Mustacone Ciperotundone Ciperenal Rotundone ␣-Ciperone Isocorimbolone Corimbolone NI

6.318 6.896 7.584 9.255 11.647 13.730 14.775 15.392 15.692 15.958 16.259 16.825 17.192 24.219 25.296 27.817 28.424 28.970 29.792 30.492 32.955 34.025 34.492 36.666 37.371 37.817 38.058 39.367 43.064 44.189 8.463 from 46.733

Total

C2

1.13 1.99

1.83 3.49

1.86 0.85

C3

C4

4.89

7.86

2.67 1.29 0.63

3.35 1.64

5.62

1.41 2.25

2.97

5.38

6.08

1.32

1.05

11.00 2.63

12.75 2.88

19.92 3.81

21.19

Hydrodistillation 10.09 4.14 5.21 3.33 1.69 7.45 3.58 0.57 0.67 0.83 5.17 4.01 0.59 1.58 1.10 1.77 2.56 0.88 0.74 2.89 0.97 0.65 8.27 2.34

5.87 0.99 5.26

10.59 5.44 15.40 44.13

12.35 5.45 15.26 37.38

18.52 4.49 12.46 25.54

19.45 5.46 18.32 10.15

22.67

100.00

100.00

100.00

100.00

100.00

NI = not identified, C1, C2, C3, C4 = experimental conditions for SFE.

Fig. 2. P–x–y diagram of CO2 (1) + priprioca extract (2).

Fig. 3. Kinetics of priprioca oil extraction obtained under different experimental conditions.

I.C.M. da Silva et al. / J. of Supercritical Fluids 88 (2014) 134–141 Table 3 Yields obtained from SFE and HDE for priprioca oil. Extraction condition

Yield (%)

SFE C1 (313 K/25 MPa) C2 (323 K/25 MPa) C3 (333 K/25 MPa) C4 (333 K/13 MPa) HDE (373 K/0.1 MPa)

3.1 ± 0.1 3.3 ± 0.2 3.3 ± 0.2 1.2 ± 0.2 0.3 ± 0.1

Table 4 Antifungal activity of priprioca extract (SFE) against germination of Cladosporium sphaerospermum ATCC 4464 spores. Experimental condition

MIC

C1 C2 C3 C4 Hydrodistillation

>1000 ␮g/mL >1000 ␮g/mL >1000 ␮g/mL =1000 ␮g/mL a Not tested

a

Not enough material for the test.

of priprioca oil extraction obtained under different experimental conditions. Table 3 shows the percentage yields of extractions for each experimental condition. The percentage yield was calculated according to the following relation: Yield (%) =

 mass extracted dry solid mass

× 100

(16)

By hydrodistillation, the volume of oil obtained was 1 mL, and by using an appropriate capillary for calculating the oil density (0.267 g/mL), one may then find the yield in mass. According to Table 3 it is possible to observe that the supercritical extraction process furnished higher yields compared to that one obtained by hydrodistillation, mainly in experiments C1, C2 and C3 (monophase) because of the facilitated mass transference and high solubility of the terpenes in carbon dioxide. Morever, no statistical differences are observed between the values for the conditions C1, C2 and C3 concerning the yields (see Table 3 and Fig. 3). In that case, the density has no influence in the final yield and, the number of phases determines the values of the yield. For conditions C1, C2 and C3, there is one fluid phase in the system and the density values do not affect the final yield of the extractions. In the monophase region, the extraction kinetic is also faster than the two-phase region (see Fig. 3). 3.3. Biological activities 3.3.1. Antifungal and antibacterial activities Tables 4 and 5 show the results of the antimicrobial activities tests for the priprioca extracts expressed in ␮g/mL. In order to classify the antimicrobial and antifungal activities for the priprioca extracts the authors employed the following criteria: the extracts presenting a MIC lower than 100 ␮g/mL Table 5 Antibacterial activity of priprioca extract (SFE) against Staphylococcus aureus ATCC 25923. Experimental condition

MIC

CBM

C1 C2 C3 C4 Hydrodistillation

a

a

a

Not tested =1000 ␮g/mL =1000 ␮g/mL >1000 ␮g/mL a Not tested

Not tested =1000 ␮g/mL =1000 ␮g/mL >1000 ␮g/mL a Not tested

Not enough material for the test. All experiments were performed in triplicate.

139

were considered as having strong antimicrobial activity; those presenting a MIC between 100 and 500 ␮g/mL were considered moderately active, a MIC between 500 and 1000 ␮g/mL were considered as having weak activity and a MIC greater than 1000 ␮g/mL were considered inactive [20]. According to the results of Table 5, one may observe that the extracts obtained in the experimental conditions C2 and C3 presented weak activity against the Gram-positive bacteria, thus confirming studies presented in the literature. The therapeutic properties of the oil have already been demonstrated by various authors, and include antibacterial activity against S. aureus and P. aeruginosa [4,5]. Based on the results of Table 4, one may note that a weak antifungal activity was observed on the extract obtained by the experimental condition C4. Based on the classification criteria adopted, the results of the afore mentioned activities did not prove to be effective, however, it is important to observe that the antibacterial activity seems to be related to the presence of terpene(s) selectively obtained by CO2. No directly correlation has already been attributed to specifically the triterpene types neither in this and nor in previous works (even isolated from other vegetal species) concerning the antibacterial or antifungal purposes. Despite that, an antiplasmodial activity has already been assigned to corimbolone and mustacone sesquiterpenes isolated from the dichlorometane extract of Cyperus articulates rhizomes [21] and, detected as major compounds in the SFE extracts. In addition, the ␣-cyperone proved to be a promising inhibitor of ␣-hemolysin (Hla) production by S. aureus and protects A549 lung cells from this bacterium. Another group [22] evaluated the antibacterial and antifungal activities of the essential oil of C. conglomerates rich in ␣ -cyperone (10.5%) against a panel of five bacterial and two fungal strains. The oil showed moderate activity against all the tested microbial strains. So it is thus justifiable to investigate the bioassay-guided fractionation of these extracts in order to analyze whether there has been an increase in the inhibition percentage according to the purification steps. 3.4. Modeling and mathematical simulation of supercritical extraction Fig. 4 presents the results of the solution for the mathematical model obtained via GITT and the experimental data from the extraction kinetics. The entry parameters (optimized) that were used in the computing code developed for solving the mathematical model via GITT in order to obtain the response variable (extracted mass) are described in Table 6. The values of the initial mass concentration in the pore volume (Cpo ) of the axial dispersion coefficient (Dax ) of the binary diffusion coefficient (Dab ) and the mass transfer coefficient (Kf ), were optimized according to the trial and error procedure, always observing the behavior of the extraction curves for a better approximation of the experimental data; and the values (of these parameters) utilized as the “initial guess” were obtained from the correlations mentioned in [23,24]. The values for Ed were obtained considering the linear behavior in the first 30 min of extraction. From Fig. 4 is observed that the mathematical model agreed better with the experimental data of case C4, where extraction takes place in equilibrium situation between phases. This result was expected already since that the mathematical model of two-phases considers the existence of equilibrium between the oil contained in the solid matrix and in internal pores existing. The equilibrium relationship Ed = Cs /Cp (where Cs is the concentration of solute in the solid matrix) is a linear relation that apparently can only be justified for the case C4. For a better applicability of the model in the conditions C1, C2 and C3 would be necessary that Ed were nonlinear function of Cp . However, is observed that the model (even with

140

I.C.M. da Silva et al. / J. of Supercritical Fluids 88 (2014) 134–141

Fig. 4. Kinetics of extraction for cases C1, C2, C3 and C4.

Table 6 Parameters used in mathematical modeling of the experimental data for the kinetic curves in supercritical extraction. Parameters

C1

C2

C3

C4

L (m) dL (m) Co Ast (m2 ) Dab (m2 /s) m (sólido seco) (kg) dp (m) Qfsc (m3 /s) Cpo (kg oil/kg CO2 ) fsc (kg/m3 ) Dax (m2 /s) t (min) ap (1/m) Ed εb εp  fsc (cpoise) Kf (m/s)

0.28560 0.285 × 10−1 0 8333.33 0.7792 × 10−3 0.763 × 10−4 0.72 × 10−3 0.50 × 10−7 0.560 × 10−1 880 0.360 × 10−4 110 6/dp 0.1760 × 10−1 0.4620 0.333 0.082 0.762 × 10−2

0.28560 0.285 × 10−1 0 8333.33 0.4994 × 10−3 0.774 × 10−4 0.72 × 10−3 0.50 × 10−7 0.620 × 10−1 835 0.234 × 10−4 120 6/dp 0.1840 × 10−1 0.4686 0.333 0.075 0.595 × 10−2

0.28560 0.285 × 10−1 0 8333.33 0.2390 × 10−3 0.809 × 10−4 0.72 × 10−3 0.50 × 10−7 0.640 × 10−1 788 0.117 × 10−4 140 6/dp 0.2050 × 10−1 0.4686 0.333 0.069 0.391 × 10−2

0.28560 0.285 × 10−1 0 8333.33 0.1943 × 10−4 0.829 × 10−4 0.72 × 10−3 0.50 × 10−7 0.328 × 10−1 507 0.975 × 10−6 120 6/dp 0.1000 × 10−1 0.5018 0.333 0.040 0.937 × 10−3

limitations) reasonably represents the experimental conditions C1, C2 and C3 (outside the region of equilibrium). In Table 6, the values of the principal parameters related to the mass transfer phenomenon (Dax , Dab and Kf ) for the extraction operating conditions in which the experiments (C1, C2, C3 and C4) were conducted demonstrates the importance of knowing the phase behaviors. In the operational condition of C4 extraction the values of Dax , Dab and Kf have at least one order of grandeur lower than the values of the other conditions. Further, it is observed that the coefficients values for Ed are constant and very close to zero, the coefficients values for Dax are not constant but are very small and the coefficients values for Cpo present growing behavior with increase temperature for C1, C2 and C3 conditions and have significant values. The behavior of the kinetic curves shown in Fig. 4 can also be understand based on knowledge of the principal parameters related to mass transfer and phase equilibrium of the system.

The extracted and accumulated mass calculated according to the following equation.

E=Q

t

y|h=H dt

(17)

0

In which E is accumulated mass and Q is solvent flow. 4. Conclusions The process for supercritical extraction of priprioca oil performed in this paper proved to be more efficient than the conventional hydrodistillation extraction process, since it presented a higher oil yield in any of the experimental conditions, being in accordance with the literature. After observing the results of the experimental extraction conditions in C1, C2 and C3, it can

I.C.M. da Silva et al. / J. of Supercritical Fluids 88 (2014) 134–141

be concluded that the extraction stages needs to be conducted in up to 50 min in order to reduce operating costs, as well as working under moderate temperature conditions in order to achieve a yield range at around 2.5%. By using the data on phase equilibrium behavior of the CO2 /priprioca extract system available in the literature as a basis to define the better extractions conditions, it has been showed a greater percentage yield in oil in the region for one phase when compared to the extraction values for the region in two phases. Weak biological activities probably correlated to the presence of terpenes selected extracted by CO2 were observed. The mathematical model investigated [13] is limited for describing the supercritical extraction process in the region outside of the phase equilibrium, in which there is the greatest yield in terms of mass and thus greater transfer of mass. Acknowledgments The authors would like to thank FAPESPA (FUNDAC¸ÃO DE APOIO A PESQUISA DO ESTADO DO PARÁ/BRASIL), CNPq, and CAPES (NANOBIOTEC) for financial support. They also thank UFPA and UEM for providing physical space and the necessary equipment for research. References [1] M.G.B. Zoghbi, G.M.S.P. Guilhon, E.H.A. Andrade, K.S.S. Vilhena, Química das espécies de Cyperus conhecidas por Priprioca, in: R.C.V. Potiguara, M.G.B. Zoghbi (Eds.), Priprioca um Recurso Aromático do Pará, MPEG, UEPA, Belém, 2008, pp. 53–76. [2] A.E.S. Rocha, As espécies de Cyperaceae Juss. conhecidas como Priprioca, in: R.C.V. Potiguara, M.G.B. Zoghbi (Eds.), Priprioca um Recurso Aromático do Pará, MPEG, UEPA, Belém, 2008, pp. 13–24. [3] M.P. Correa, Dicionário das plantas úteis do Brasil e das exóticas cultivadas, Imprensa Nacional, Rio de Janeiro, 1987, pp. 35–110. [4] E. Mongelli, C. Desmarchelier, J. Coussio, et al., Antimicrobial activity and DNA interaction in medicinal plants from the Peruvian Amazon, Revista Argentina de Microbiología 27 (1995) 199–203. [5] C. Desmachelier, E. Mongelli, J. Coussio, G. Ciccia, Studies on the cytotoxicity, antimicrobial and DNA-binding activities of plants used by the Ese’ejas, Journal of Ethnopharmacology 50 (1996) 91–96. [6] E. Ngo Bum, M. Schmutz, C. Meyer, A. Rakotonirina, M. Bopelet, C. Portet, A. Jeker, S.V. Rakotonirina, H.R. Olpe, P. Herrling, Anticonvulsant properties of the methanolic extract of Cyperus articulatus (Cyperaceae), Journal of Ethnopharmacology 76 (2001) 145–150.

141

[7] C. Desmachelier, M. Repetto, J. Coussio, S. Llesuy, G. Ciccia, Total reactive antioxidant potential (TRAP) and total antioxidant reactivity (TAR) of medicinal plants used in Southwest Amazon (Bolívia and Peru), International Journal of Pharmacognosy 35 (1997) 288–296. [8] M.S. Abubakar, E.M. Abdurahman, A.K. Haruna, The repellant and antifeedant properties of Cyperus articulatus against Trilobium casteneum Hsbt, Phytotherapy Research 14 (2000) 281–283. [9] L.S. Moura, R. Favareto, P.F. Leal, M.L. Corazza, L. Cardozo Filho, M.A.A. Meireles, Phase equilibrium measurements for CO2 + priprioca extract at high pressures, The Journal of Supercritical Fluids 48 (2009) 126–130. [10] Normas Analíticas do Instituto Adolfo Lutz. v. 1, Métodos Químicos e Físicos para Análise de Alimentos, 3rd ed. IMESP, São Paulo, 1985, pp. 42–43. [11] A.S. Foust, C.W. Clump, L. Maus, L.B. Andersen, Principles of Unit Operations, Wiley International Edition, New York/Tokyo, 1960. [12] S. Angus, B. Armstrong, K.M. Reuck (Eds.), International Thermodynamic Tables of the Fluid State. Carbon Dioxide, Pergamon Press, 1976. [13] R.M. Cotta, M.D. Mikhailov, Heat Conduction – Lumped Analysis, Integral Transforms, Symbolic Computation, John Wiley & Sons, England, 1997. [14] R.M. Cotta (Ed.), The Integral Transform Method in Thermal and Fluids Science and Engineering, Begell House, Inc, New York, 1998. [15] R.M. Cotta, M.D. Mikhailov, Hybrid methods and symbolic computations, in: W. Minkowycz, E.M. Sparrow, J.Y. Murthy (Eds.), Handbook of Numerical Heat Transfer, 2nd ed., John Wiley & Sons, 2006 (Chapter 16). [16] L.A. Sphaier, R.M. Cotta, C.P. Naveira-Cotta, J.N.N. Quaresma The, UNIT algorithm for solving one-dimensional convection-diffusion problems via integral transforms, International Communications in Heat and Mass Transfer 38 (2011) 565–571. [17] National Committee for Clinical Laboratory Standards – NCCLS, Reference Method for Broth Dilution Antifungical Susceptibility Testing of Yeasts. Approved Standard M27-A, NCCLS, Wayne, PA, 1997. [18] National Committee for Clinical Laboratory Standards – NCCLS, Performance Standards for Antimicrobial Susceptibility Testing: eight international supplement M100-S14, NCCLS, Wayne, PA, 2004. [19] M.G.B. Zoghbi, E.H.A. Andrade, J. Oliveira, L.M.M. Carreira, G.M.S.P. Guilhon, Yield and chemical composition of the essential oil of the stems and rhizomes of Cyperus articulatus L. cultivated in the State of Pará, Brasil, Journal of Essential Oil Research 18 (2006) 10–12. [20] F.B. Holetz, G.L. Pessini, N.R. Sanches, D.A.G. Cortez, C.V. Nakamura, B.P. Dias Filho, Screening of some plants used in the Brazilian folk medicine for the treatment of infectious diseases, Memórias do Instituto Oswaldo Cruz 97 (2002) 1027–1031. [21] G.M. Rukunga, F.W. Muregi, S.A. Omar, J.W. Gathirwa, C.N. Muthaura, M.G. Peter, M. Heydenreich, G.M. Mungai, Anti-plasmodial activity of the extracts and two sesquiterpenes from Cyperus articulates, Fitoterapia 79 (2008) 188–190. [22] M. Luo, J. Qiu, Y. Zhang, J. Wang, J. Dong, H. Li, B. Leng, Q. Zhang, X. Dai, X. Niu, S. Zhao, X. Deng, ␣-Cyperone alleviates lung cell injury caused by Staphylococcus aureus via attenuation of ␣-hemolysin expression, Journal of Microbiology and Biotechnology 22 (2012) 1170–1176. [23] C.S. Tan, D.C. Liou, Axial dispersion of supercritical carbon dioxide in Packed Beds, Industrial & Engineering Chemistry Research 28 (1989) 1246–1250. [24] N. Wakao, S. Kaguei, Heat and Mass Transfer in Packed Beds, Gordon and Breach, New York, 1982.