Marigold (Calendula officinalis L.) oleoresin: Solubility in SC-CO2 and composition profile

Marigold (Calendula officinalis L.) oleoresin: Solubility in SC-CO2 and composition profile

Chemical Engineering and Processing 46 (2007) 99–106 Marigold (Calendula officinalis L.) oleoresin: Solubility in SC-CO2 and composition profile Leand...

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Chemical Engineering and Processing 46 (2007) 99–106

Marigold (Calendula officinalis L.) oleoresin: Solubility in SC-CO2 and composition profile Leandro Danielski a , Luanda M.A.S. Campos a , Louisiane F.V. Bresciani b , Haiko Hense a , Rosendo A. Yunes b , Sandra R.S. Ferreira a,∗ a

EQA-CTC/UFSC, Chemical Engineering and Food Engineering Department, Federal University of Santa Catarina, C.P. 476, CEP 88040-900, Florian´opolis, SC, Brazil b QMC-UFSC, Chemistry Department, Federal University of Santa Catarina, Florian´ opolis, SC, Brazil Received 21 December 2005; received in revised form 22 March 2006; accepted 7 May 2006 Available online 12 May 2006

Abstract The solubility of marigold (Calendula officinalis L.) oleoresin in supercritical CO2 (SC-CO2 ) and the composition profile of the extracts obtained using different extraction methods were investigated. Supercritical experiments were performed at different temperature (20–40 ◦ C) and pressure (120–200 bar) levels. The oleoresin solubility in SC-CO2 was determined through the dynamic method using low solvent flow rates (0.79–1.67 g CO2 /min), by assuming that the solvent was saturated downstream the extractor. The solubility varied from 4.74 × 10−4 to 17.04 × 10−4 g oleoresin/g CO2 and was correlated using the density-based equation proposed by Chrastil. The composition profile of the marigold oleoresins obtained with supercritical fluid extraction (SFE) and with organic solvent extraction using n-hexane, dichloromethane and butanol, were evaluated and compared. The results have shown differences in the composition profiles obtained from the marigold cultivated in Brazil (this work), compared to the ones from Europe culture (literature results). © 2006 Elsevier B.V. All rights reserved. Keywords: Marigold (Calendula officinalis L.); Supercritical fluid extraction; Organic solvent extraction; Composition; Solubility

1. Introduction Chemical and pharmacological studies involving medicinal plants have increased in the last decades, not only related to the isolation of active principles, but also to the characterization of new components with therapeutic activity and nutraceutic characteristics, important for the use in food industries, as well as in cosmetology and pharmacology. Calendula officinalis L. (Asteraceae), a bright yellow and orange flower, commonly known as marigold or cultivated marigold, is an annual herbaceous plant, native of Mediterranean countries. The flower is normally used as food additive to con-

Abbreviations: CER, constant extraction rate period; DCM, dichloromethane; GC, gas chromatography; GC–MS, gas chromatography–mass spectrometry; OEC, overall extraction curve; OSE, organic solvent extraction; RT, retention time in the chromatographic column; SFE, supercritical fluid extraction ∗ Corresponding author. Tel.: +55 48 3331 9448; fax: +55 48 3331 9687. E-mail address: [email protected] (S.R.S. Ferreira). 0255-2701/$ – see front matter © 2006 Elsevier B.V. All rights reserved. doi:10.1016/j.cep.2006.05.004

fer both color and flavor to foods [1,2]. Marigold is also widely used in traditional and homeopathic medicine as infusions and ointments prepared with its petals. It presents several therapeutic activities, such as anti-inflammatory, antitumorogenic and cicatrizing ones [1–4]. Antiviral and immunostimulating effects have also been reported: potential therapeutic activity against the human immunodeficiency virus (HIV) was initially observed with the in vitro use of marigold extracts to inhibit significantly the HIV-1 virus replication [3]. Several studies involving marigold extracts have been performed, mainly related to the characterization of extracts obtained with organic solvents. The results indicate that the therapeutic characteristic of its extract is partially due to the terpene content, and the most important compounds are triterpenoids, flavonoids, essential oils and sesquiterpenes. Additionally, several components from the marigold flowers have been identified ´ [5–7]. The recent work published by Cetkovi´ c et al. [8] dealt with the antioxidant properties of marigold extracts obtained from two different species, the growing wild marigold (Calendula arvensis L.) and cultivated marigold (C. officinalis L.). Water

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extracts of cultivated marigold provided the best antioxidant properties, determined according to total phenolic and flavonoid contents, which present high interest in food industries as well as in phytotherapy. The limitations of the conventional extraction methods, such as time and solvent consuming aspects, increase the interest towards alternative processes such as supercritical fluid extraction (SFE). This high-pressure operation has become a reliable technology in the last decades, particularly for the extraction of components from medicinal plants, due to its unique characteristics, such as mild operational conditions and the achievement of extracts with high purity [9,10]. The extraction rate from solid materials, such as roots, leaves, seeds and other vegetable parts, is often defined by the solubility of these complex mixtures within the SC-CO2 phase. SFE processes optimization requires the knowledge of phase equilibrium (solubility data), which contributes to the selection of the operating conditions [9,11]. In the last years, several studies evaluating experimental solubility data for different solutes and complex mixtures in SC-CO2 have been correlated by using the so-called density-based equations. The results, associated to the use of equations such as the one represented by Chrastil’s model [12], are extensively presented in the literature [13–15]. Therefore, the aim of this study was to obtain solubility experimental data of marigold (C. officinalis L.) oleoresin in SC-CO2 and correlate the experimental results with the densitybased equation proposed by Chrastil [12]. Also, the yield and the composition profile of marigold oleoresin obtained in SFE was evaluated and compared with low-pressure extraction using n-hexane, dichloromethane (DCM) and butanol as solvents. The composition of marigold oleoresin was additionally compared with literature results. 2. Materials and methods 2.1. Marigold flowers Marigold flowers were purchased from Chamel Ind. & Com. (Campo Largo, PR, Brazil), which certified the authenticity of the plant material as the Brazilian commercialized C. officinalis L. The dried plant was stored at −10 ◦ C in a domestic freezer. The flowers were grounded immediately before the extractions in a domestic coffee grinder (Melitta, SP, Brazil) and the particle size was determined as an average particle diameter of 0.62 mm [16]. 2.2. Apparatus SFE experiments were carried out in a high-pressure unit, as described by Michielin et al. [17]. CO2 was supplied by White Martins, Brazil (99.9% purity). The solvent was delivered into a surge tank where the temperature was kept at 1.0 ◦ C (±0.1 ◦ C) by a thermostatic bath (Microqu´ımica, Model MQBTZ99-20, SC, Brazil) in order to guarantee liquid CO2 in the pump head section. The solvent was then pressurized by a high-pressure pump (Thermo Separation Products, Model 3200 P/F, Fremont, CA, USA), up to the operational pressure. A jacked stainless

steel extraction column, with 40.0 cm length and 2.1 cm i.d. (Suprilab, SP, Brazil), was connected downstream the pump, with the temperature controlled by a thermostatic bath. Dense CO2 was expanded through a micro-metering valve (Autoclave Engineers, Model 30VRMM4812, PA, USA) connected downstream the extractor. The solute (marigold oleoresin) was collected in amber flasks connected to a calibrated wet-test meter (LATESC/EQA-UFSC), used to measure the CO2 flow rate and consume. A fixed bed was formed with 40.0 g (±0.1 g) of grounded marigold flowers, placed inside the extraction vessel. The operating conditions were adjusted and a static period of 3 h before the extraction was observed. Samples were collected at preestablished time intervals up to 64 h extraction. The experiments were carried out at temperatures from 20.0 to 40.0 ◦ C (±0.1 ◦ C) and pressures from 120.0 to 200.0 bar (±2.5 bar). 2.3. Solubility determination The solubility results were obtained using the dynamic method of extraction and calculated from the slope of the linear portion of the overall extraction curves (OEC) performed at equilibrium conditions, i.e., at low solvent flow rate to allow the solvent saturation with solute. The linear part of the curves indicates the constant extraction rate period (CER) [18]. The OEC are represented by solvent rate (solvent/solid ratio) versus extraction rate (solute/solid ratio) and, in order to obtain the solubility values, two straight lines were fitted to the OEC. The first line represented the CER and the second one, the decreasing extraction rate. When the curve reached a fixed plateau, it was assumed that the maximum yield of oleoresin was achieved. A statistical analysis was performed to evaluate the best fit to the experimental results by using the procedures PROC REG and PROC NLIN of SAS for Windows 6.11 [19]. The slope of the CER period was calculated and the results were expressed by YCTE , the solute concentration in the solvent phase (g oleoresin/g CO2 ) [15,16,18]. 2.4. Solubility data correlation Empirical models for solubility determination are advantageous since they discharge the use of physical–chemical properties, normally difficult to obtain. The density-based equation proposed by Chrastil [12] considers a solvate-complex between solute and SC-CO2 at equilibrium condition. The model is a linear relation between solute solubility, solvent density and the system temperature, as presented by the following equation (Eq. (1)): ln Y ∗ = k ln d +

a +b T

(1)

where Y* , d and T represent the solute solubility (g/l) in the supercritical solvent, the CO2 density (kg/m3 ) and the process temperature (◦ C), respectively. The constant a corresponds to temperature effect, while constant b is related to solute and solvent physical properties. Moreover, k corresponds to the number of solvent molecules that create a complex with one single

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molecule of the solute. The constants a, b and k are adjustable parameters of the Chrastil’s model. 2.5. Organic solvent extractions (OSE) To avoid thermal degradation of thermolabile components from marigold oleoresin, the extracting conventional method consists of cold maceration of marigold flowers. The extraction was performed with dried flower powder (104.0 ± 0.1 g) placed in methanol for 5 days. The extract was then evaporated under reduced pressure up to 10–20% of the initial volume to obtain the crude extract (CE). The CE was partitioned with different solvents, from non-polar to polar ones: n-hexane, dichlormethane (DCM) and butanol. The corresponding volumes of the organic solvents were: n-hexane (600 ml), DCM (450 ml) and butanol (800 ml). 2.6. Extract components identification Gas chromatography (GC) analyses were performed by a gas chromatograph (Shimadzu, Model CGS-14A, Kyoto, Japan) equipped with a 25 m × 0.25 mm i.d. coated column (0.3 ␮m film thickness) with cross-linked polymethylsiloxane as stationary phase (column LM-1, L&M, SP, Brazil). The samples were admitted using a split-mode injection (split rate at 1:20) and the analyses were run with 1 ␮l extract injection diluted with DCM at 1 ml. The temperature of the flame ionization detector (FID) was kept at 320 ◦ C and the injector’s temperature was 280 ◦ C. The column temperature program started at 40 ◦ C (8 ◦ C/min rate up to 310 ◦ C) and was held for 10 min. Hydrogen was used as carrier gas at 2 ml/min flow rate. The area peak of the chromatograms was integrated using the program Cromatografia (Microqu´ımica, Brazil). The components of the marigold oleoresin were identified by gas chromatography–mass spectrometry analysis—GC–MS (Shimadzu, Model GCMS-QP2000A, Kyoto, Japan). The temperature programs and the columns used in GC and in GC–MS analyses were the same. The identification of the oil components was based on a database for natural products (NIST Mass-Spectral Library with Windows search program-V2), where GC–MS results were compared. Reported data from marigold constituents were also used to identify the extract components [20,21]. 3. Results and discussions 3.1. Determination of the solubility data In order to use the dynamic method to assess experimental solubility values, the fixed bed was considered long enough and the solvent flow rate sufficiently low to warranty adequate contact time between phases, assuring the solvent saturation. The effect of solvent flow rate on the solubility determination of marigold oleoresin with supercritical CO2 is presented in Fig. 1 for assays performed at 200 bar and three different temperatures (26, 30 and 40 ◦ C). The results indicate the value of the slope of the straight line adjusted to the CER period, also called YCTE , for extractions performed at various flow rates. From Fig. 1

Fig. 1. Effect of the solvent flow rate on YCTE value for marigold oleoresin at 200 bar.

we observed that the highest values of YCTE for each temperature were obtained near 1.10 g CO2 /min (±0.01 g CO2 /min). At this condition, it was assumed the solvent saturation with the oleoresin. At lower solvent flow rates, the YCTE decreases, probably due to the higher intra-particle solid diffusion, which may overcome the convective effects [15,18,22]. Thus, for the set of experiments evaluated in this work, the solubility (Y* ) for each operational condition was considered as the highest value of YCTE . Table 1 shows the solvent flow rate, the CO2 density and YCTE values for the conditions of temperature and pressure studied, indicating a solvent density varying from 719.1 (120 bar, 40 ◦ C) to 910.2 kg CO2 /m3 (200 bar, 26 ◦ C). The CO2 density was obtained according to Angus et al. [23] and the YCTE were determined from the slope of the linear portion of the OEC. Fig. 2 shows OEC at 120 and 200 bar (40 ◦ C). The curves show the typical behavior of the supercritical extraction curves: the linear portion or CER, where the YCTE values were calculated, Table 1 Supercritical fluid extraction of marigold oleoresin: experimental conditions (temperature, pressure, density, solvent flow rate) and YCTE values T (◦ C)

P (bar)

26 26 26 26 26 30 30 30 30 30 40 40 40 40 40 40 40 40

120 150 200 200 200 120 150 150 200 200 120 150 150 200 200 200 200 200

dsolvent (kg/m3 ) 839.1 871.5 910.2 910.2 910.2 809.7 847.8 847.8 890.9 890.9 719.1 781.3 781.3 841.5 841.5 841.5 841.5 841.5

Qsolvent (g CO2 /min)

YCTE (×104 ) (g oleoresin/g CO2 )

1.10 1.10 0.90 1.08 1.34 1.10 1.10 1.30 0.90 1.10 1.10 1.21 1.67 0.79 0.94 1.10 1.33 1.57

11.75 11.44 10.45 13.77 10.42 9.93 6.15 5.75 10.90 11.17 6.57 4.74 6.98 7.22 11.15 17.04 11.91 11.54

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Fig. 2. Overall extraction curves (OEC) for the supercritical fluid extraction of marigold oleoresin at 40 ◦ C.

and the falling extraction rate period. The pressure influence is represented by the increase in the solute extracted due to the increase in solvent density (from 719.1 to 937.5 kg/m3 ). The supercritical fluid (SF) presents liquid-like density, increasing with pressure and decreasing with temperature. When increasing the solvent density, the solubility increases, enhancing the number of soluble components and decreasing selectivity [11]. The solubility data (Y* ) for marigold oleoresin are also presented in Table 1 (bold face). Higher solubility values were achieved at higher solvent densities, where the number of soluble components increased due to the enhancement of SC-CO2 solvating power. According to Table 1, the Y* values increased with increasing pressure at constant temperatures, except for 30 ◦ C, where solubility decreases at 150 bar. This behavior could be justified by the vicinity of the critical temperature, where few changes in the process conditions result in remarkable effect in the solute solubilization. The temperature effect is more complex as can be observed in the solubility isotherms in Fig. 3. The figure shows the crossing of the isotherms, indicating the cross-over phenomena. This inversion pattern was observed near 140 bar, the cross-over pressures, for 30 and 40 ◦ C and close to 160 bar, for 26 and 40 ◦ C. The cross-over behavior is due to the competing effects of reducing solvent density and increasing solute vapor pressure with temperature increase [17,24–27]. The difference in cross-over pressure, for the isotherms studied, is probably due to the lowest influence of solute vapor pressure

Fig. 4. Marigold solubility data in supercritical CO2 : comparing experimental and modelled results using Chrastil’s equation. Table 2 Data correlation for horsetail oleoresin solubility: parameters for Chrastil’s equation and standard deviations (STD) Parameter values k a b

2.69 7204.08 −42.24

STD for isotherm data 26 ◦ C 30 ◦ C 40 ◦ C

11% 22% 73%

over density effect at 26 ◦ C, therefore extending the region influenced by the solvent density effect, up to 160 bar, if compared with other isotherms, where the crossing was near 140 bar. Fig. 4 shows the correlation of the experimental solubility data of marigold oleoresin using the empirical model presented by Chrastil. The solubility isotherms are presented at 26, 30 and 40 ◦ C and the adjusted parameters for Eq. (1) were obtained from the experimental curve correlation and are shown in Table 2 with the values for standard deviation (STD). The results presented in Fig. 4 and Table 2 indicate good agreement between experimental and modeled data for 26 ◦ C (highest density) for all pressures studied, with STD at 11%. At 30 ◦ C the adjustment quality decreases, with a STD of 22%, and at 40 ◦ C the correlated results did not fit well to experimental data, probably because the experiments were carried on the vicinity of the crossover region (near 140 bar) and/or the critical point (150 bar and 30 ◦ C). At 26 ◦ C, the effect of solvent density in the solubilization was predominant over the effect of solute vapor pressure, and the Chrastil’s model is strongly dependant on the solvent density. 4. Extraction yield evaluation for marigold extracts

Fig. 3. Pressure and temperature effects on solubility of marigold oleoresin on supercritical CO2 .

Organic solvent extraction (OSE) with n-hexane, DCM and butanol are compared in Table 3, in terms of extracting yield, with SFE performed at 20–40 ◦ C and 120–200 bar. The sol-

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Table 3 Marigold oleoresin extraction yield: organic solvent extraction and SFE at different extracting conditions Organic solvent extraction Solvent

Supercritical fluid extraction Yield, w/w (%)

n-Hexane

6.94

Dichloromethane

22.40

Butanol

21.90

Conditions (bar/◦ C)

Qsolvent (g CO2 /min)

Yield, w/w (%)

120/20 120/26 120/30 120/40 150/26 150/40 200/20 200/26 200/40

2.79 1.10 1.10 2.05 1.10 1.67 3.00 2.98 2.96

2.63 2.32 2.08 2.12 2.50 3.07 3.31 3.14 3.54

vent flow rate for SFE assays (Table 3) varied from 1.1 to 3.0 g CO2 /min, and, to compensate that difference, the yield was obtained at close values of consumed CO2 . Therefore, the SFE yield results indicate a behavior similar to the Y* (Fig. 3), with cross-over pressures near 150 bar for isotherms at 20 and 40 ◦ C. The lowest OSE yield was obtained with n-hexane, a nonpolar solvent, indicating that marigold contains rather polar than non-polar components. The highest yield, obtained with DCM (22.4%, w/w), was probably caused by the extraction of components like waxes, chlorophyll and others, not detected by the GC analysis. The SFE yield was up to 3.54%, w/w (±0.02%, w/w), comparable in magnitude order with n-hexane results. This behavior is justified by the non-polar characteristic of CO2 , representing the solubilization of preferably non-polar components. The results also suggest the use of DCM and butanol as co-solvents for SFE with the purpose of improve the extraction yield, although, careful investigation in the composition should be performed to validate the process. The comparison between SFE and OSE indicates low efficiency of the CO2 extraction due to the non-polar character of the solvent, if compared with DCM and butanol.

normally as an anabolic steroid. Then, the use of high-density solvents to obtain marigold extract is justified by the complex character of the oleoresin and the presence of high molecular weight substances (∼300 kg/kg mol) [10,20,28,29]. In order to compare the SFE with OSE in terms of composition, the OSE fractions with n-hexane and DCM (nonpolar solvents) were evaluated through GC analysis. The use of GC/GC–MS analysis (for volatile components) is justified from the interest in the long-chain hydrocarbons and fatty acids present in the marigold extract. In Table 4 it is observed that, only components 6 (0.62% p.a.), 7 (1.15% p.a.), 8 (1.41% p.a.), 12 (2.78% p.a.), 13 (21.80% p.a.), 24 (11.94% p.a.) and 25 (31.59% p.a.) for DCM and 6 (1.81% p.a.), 7 (2.38% p.a.), 9 (20.43% p.a.), 13 (8.79% p.a.) and 25 (3.76% p.a.) for n-hexane, were detected in the OSE extracts. This behavior indicates a remarkably poor component variety compared with the SFE extracts. Therefore, besides the highest yield of the DCM fraction, the composition analyses indicate that the main target components, such as guaiol and taraxasterol, were found mostly in the supercritical samples.

4.1. Composition profile of marigold extracts Fig. 5 shows the result of the GC–MS analysis for a SFE at 20 ◦ C and 188 bar (930.4 kg CO2 /m3 ). The identified components, the molecular weight and extract composition are listed in Table 4 (peak numbers according to Fig. 5) for different extracts: OSE (DCM and hexane) and SFE (40 ◦ C and at 120, 150 and 188 bar). Table 4 indicates that marigold extracts are rich in non-polar components as a result of the solvents characteristic and also to the range of the GC analysis used in this work. The extracting conditions avoid the presence of glycoside flavonoids and high molecular weight substances (above 500 kg/kg mol), unlike the Hamburger et al. [2] results, that show a marigold extract composed mainly by triterpenoid esters. The SFE marigold oleoresins present viscous and brownish aspect and are mainly formed by a mixture with diverse applications in food industries and phytotherapy. Components listed in Table 4 are related to several uses: acetyl eugenol, phenol4-octyl, cedrol and guaiol are essential oils; nonadecane and eicosane are pheromones and taraxasterol is a triterpenoid used

Fig. 5. GC–MS result for marigold oleoresin obtained with SFE at 20 ◦ C and 188 bar.

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Table 4 Marigold oleoresin integrated composition for extracts obtained with organic solvents (DCM and n-hexane) and CO2 at 40 ◦ C and different pressures (120, 150, 188 bar) Peak

1 2 3 4 6 7 8 9 10 11 12 13 14 15 16 17 19 20 21 22 23 24 25 NIa a

Component

Mol (kg/kg mol)

Acetyl eugenol Phenol-4-octyl Guaiol Cedrol Octadecane Tetradecanoic acid Nonadecane Eicosane Heneicosane Docosane Tricosane Tetracosane Pentacosane Hexacosane Heptacosane Octacosane Eicosane-7-hexyl Eicosane-9-octyl Canescegenine Cholest-4-en-3-one-14-methyl Taraxasterol 1-Octadecanol 1,16-Hexadecanediol

206 206 222 222 254 228 268 282 296 310 324 338 352 366 380 394 366 394 420 398 426 270 258 –

DCM

n-Hexane

120 bar

150 bar

188 bar

– – – – 0.62 1.15 1.41 – – – 2.78 21.80 – – – – – – – – – 11.94 31.59 28.70

– – – – 1.81 2.38 – 20.43 – – – 8.79 – – – – – – – – – – 3.76 62.84

– – – – – 0.76 0.40 0.56 – 0.60 0.50 0.11 – 10.70 0.90 12.74 2.75 – 0.33 0.37 0.21 – – 69.09

– – – – – – 0.32 3.43 0.32 – – 9.76 – – 0.52 11.98 0.18 1.27 0.13 0.12 0.13 – – 66.38

7.44 1.07 5.07 1.94 4.14 0.95 0.48 3.06 0.21 4.88 0.50 10.47 1.04 18.23 1.44 9.95 0.45 0.61 0.79 0.83 1.19 0.15 0.26 26.23

NI: Non identified.

Results from Table 4 indicate practically no changes in the composition profile of supercritical extracts obtained at 120 and 150 bar. Although, increasing pressure up to 188 bar, the number of identified components increases, reducing the process selectivity. By increasing pressure, the concentration of high molecular weight components also increases. This behavior is a result of the solvent density effect, i.e., high-density indicates low selectivity and high solvating power [17]. In SFE, the extracting time also affects the oleoresin composition. Table 5 compares two fractions of a SFE obtained at 20 ◦ C, 200 bar and 1.65 l CO2 /min, in terms of compositions characteristics. One extract collected up to 100 min extraction and other from 100 to 360 min. The results are expressed in terms of number of substances, the components retention time Table 5 Number of components evaluate for marigold oleoresin obtained with SFE at 20 ◦ C, 200 bar and different extracting periods (100 and 360 min) Observationa

100 min

360 min

Component number Number of components with RT lower than 25 min Number of components with RT higher than 25 min Relative concentration of components with RT lower than 25 min (in %) Relative concentration of components with RT higher than 25 min (in %)

20.0 11.0

10.0 2.0

9.0

8.0

30.3

7.2

69.7

92.8

a

% Peak area

RT = Retention time in the chromatographic column.

(RT) in the chromatographic column and the relative area peak of the chromatograms. At the beginning of extraction (fraction up to 100 min), the oleoresin show a larger variety of components, compared to the second fraction. This behavior indicates the exhaustion of some components during extraction. It is also observed that the hydrocarbon sequence (peaks 8–20 in Table 4), which presents RT higher than 25 min, is preferably extracted in the late fraction, after the lower molecular weight components are extinguished. The composition profile of the marigold oleoresin from a Brazilian harvested plant (this work; Table 4) was compared with literature data obtained from European harvested plant [7,21,30]. The results show significant difference in the identified components. From the Brazilian cultivated plant (present work), the identified components were mostly aliphatic hydrocarbons, like tetracosane, hexacosane and octacosane; triterpenes, like canescegenine, cholest-4-en-3-one-14-methyl and taraxasterol; and alcohols, like 1-octadecanol (high molecular weight substances). Literature data, representing European cultivated flowers, specify the presence of triterpenes, like longispinogenine, heliantriol C, helianol, ␣-amirin and ␺taraxasterol [1,21,30]. Unlike these results, Zitterl-Eglseer et al. [7], indicate triterpenoids as the main components in marigold, conferring anti-inflammatory activity to the extract. The Brazilian harvested plant is commercialized in Brazil as C. officinalis L. The differences detected in the extract composition, compared with literature data, are justified by the diversities in the raw material (seasoning, harvest, soil, etc.) and mainly due to the extraction technique used to obtain the marigold ole-

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oresin. Also, according to literature, the use of higher pressures for the CO2 extraction (up to 500 bar) allows the extraction of triterpenoid esters, such as faradiol 3-O-laurate, palmitate and myristate [2], not detected at the conditions studied in this work. An important result of the present research is the identification of the homologous series of hydrocarbons, such as components 6 and 8–20, as presented in Table 4. According to the literature, the hydrocarbon fraction was never considered a significant part of marigold extracts, but the present work point to the consideration of this important class of substance [7,20,21,29,30]. 5. Conclusions The high-pressure extraction is a powerful technology to obtain marigold extract, and a density-based correlation used to evaluate the solubility in SC-CO2 is most adequate for low temperatures. Experimental solubility isotherms represent well the cross-over region, pointing the importance of solute vapor pressure and solvent density to describe the temperature effect for solubility. Although the yield obtained with supercritical CO2 is lower than the conventional solvent, it is important to observe the selectivity of the process through the oleoresin composition. The composition profile of the extract varies slightly with the CO2 density, but present pronounced differences from the conventional extracts. To study the pharmacological potential of the extract, further evaluation of the supercritical extract must be accomplished, especially to investigate the process selectivity. Conventional organic solvents can also be used as co-solvents in the SFE due to its effect related to the polarity, as well as selectivity, on the solvent mixture characteristics. Only three triterpenoids were identified in the extracts obtained from plants harvested in Brazil (Table 4), different from the extracts obtained from European plant, as presented in the literature [7,22,30]. The Brazilian extract shows a rich hydrocarbon homologous series (13 components) and a triterpenic mono-alcohol such as taraxasterol. The differences in the composition profile between the extracts from Brazilian and European plants may indicate the influence of the harvest conditions, the identification technique used and mostly extraction technique [30]. Acknowledgements The authors wish to acknowledge CAPES (Coordenac¸a˜ o de Aperfeic¸oamento de Pessoal de N´ıvel Superior, Brazil) and CNPq (Conselho Nacional de Desenvolvimento Cient´ıfico e Tecnol´ogico, Brazil) for the financial support. Appendix A. Nomenclature a b d

constant correspondent to temperature effect in Chrastil’s model constant related to solute and solvent physical properties in Chrastil’s model CO2 density (kg/m3 )

k Q T Y* YCTE

105

number of solvent molecules that create a complex with one single molecule of solute, in the Chrastil’s model solvent flow rate (g CO2 /min) process temperature (◦ C) solute solubility in the supercritical solvent (g oleoresin/g CO2 ) slope of the linear portion of the extraction curve (g oleoresin/g CO2 )

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