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J. of Supercritical Fluids 44 (2008) 308314

Process optimization and extraction rate analysis of carotenoids extraction from rosehip fruit using supercritical CO2
Siti Machmudah, Yukari Kawahito, Mitsuru Sasaki, Motonobu Goto
Graduate School of Science and Technology, Kumamoto University, Kurokami 2-39-1, Kumamoto 860-8555, Japan Received 26 February 2007; received in revised form 31 August 2007; accepted 11 September 2007

Abstract Carotenoids have been extracted from rosehip fruit using supercritical CO2 at various extraction conditions. Carotenoids content in the extract was also determined by high-performance liquid chromatography (HPLC) method. Extraction was carried out at pressures of 150450 bar, temperatures of 4080 C and CO2 ow rates of 24 ml/min. A full 33 factorial design, in which pressure, temperature and CO2 ow rate were used as design factors, coupled with statistical analysis of the results, by using analysis of variance (ANOVA), was used to optimize operating condition of carotenoids extraction in SC-CO2 . Extraction rate constant was calculated from the dependence of extraction yield on extraction time at 10, 20, 30, 50, 80, 110 and 150 min. Based on the experimental results, the total carotenoids extracted from rosehip fruit were found to be 10.3520.88 mg/g fruit, and the maximum amount of carotenoids extracted was obtained at 80 C, 450 bar and 4 ml/min. As determined by HPLC methods, the carotenoids extracted contained lycopene and -carotene as main components, and small amount of lutein. The amount of lycopene, -carotene and lutein extracted were 1.18014.37 mg/g feed, 0.1541.017 mg/g feed and 1.25816.84 g/g feed, respectively. The optimization results demonstrated that temperature was to be the inuential variable on the extraction yield of carotenoids, with the statistical signicant effect p-value was smaller than 0.05. For lycopene extracted, all variables (temperature, pressure and CO2 ow rate) were to be the inuential variables, but there is no possible interaction among variables. For -carotene and lutein extracted, the inuential variables were pressure and CO2 ow rate without any interactions among variables. The mean extraction rate constant, k, of total carotenoids increased with increasing pressure and temperature, and the k values were (3.3445.851) 103 min1 . 2007 Elsevier B.V. All rights reserved.
Keywords: Carotenoid; Extraction rate constant; Rosehip; Supercritical CO2 extraction

1. Introduction The reddish orange colored fruit of rosehip (Rosa canina) is widely used for medical purposes. A valuable ingredient of this fruit, vitamin C [1], is used for the prevention of disease in the form of rosehip tea. In addition, rosehip contains biological active compounds, such as carotenoids [2], tocopherol [3], polyphenolics [4], amino acids, fruit acids and tanning substances. Carotenoids are widespread pigments in plants in which they are involved in photosynthesis and photoprotection [5], but they are also found in animal tissues where they may act as antioxidants or as immunomodulating, antimutagenic and tumor-preventing agents. The application of carotenoids in medicine and cosmetics are well documented as is their utilization as food additives (colorants and antioxidants).

Corresponding author. Tel.: +81 96 342 3664; fax: +81 96 342 3679. E-mail address: mgoto@kumamoto-u.ac.jp (M. Goto).

Supercritical uid extraction is an environmentally benign alternative to conventional industrial solvent extraction, with the important advantage of giving products that are completely free from toxic residues. One of the most frequently used supercritical uids is carbon dioxide, because it is neither toxic nor ammable, and also available at low cost and high purity. Because of its moderate critical temperature, CO2 can be used to extract thermally labile compounds. Taking into account these characteristics, CO2 is an ideal solvent in food, dye, pharmaceutical and cosmetic industries. Previous work on SC-CO2 extraction of carotenoids-containing rosehip fruits has been reported by Illes et al. [6], but the inuence of extraction conditions on the total carotenoids and carotenoids composition have not been investigated so far. They reported that the major carotenoids were lycopene, -carotene and lutein. For development of extraction process, employment of rosehip extraction with SC-CO2 implies the necessity for optimization of the variables involved in SC-CO2 extraction process, i.e. temperature, pressure and CO2 ow rate. To optimize the

0896-8446/$ see front matter 2007 Elsevier B.V. All rights reserved. doi:10.1016/j.supu.2007.09.032

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extraction processes and uncover the mass transfer models, the extraction kinetics is denitely necessary. However, the kinetics of carotenoids extraction from rosehip fruit has not yet been reported. Furthermore, the work presented here involved taking the experimental data and carrying out a full factorial design in order to analyze the effect of above-mentioned variables on the extraction yield of carotenoids. By using analysis of variance (ANOVA), the best extraction conditions for the studied range of parameters were found and the inuential variables on the extraction yield were obtained. Based on the experimental and extraction kinetics data, extraction rate constant of total carotenoids extracted was determined. 2. Materials and methods 2.1. Materials and chemicals Sample of dry rosehip (fruits including seeds) was obtained from Sanoore Co., France. Prior to processing, the dry rosehip fruits were separated from the seeds. The fruits (seeds free) were ground in a coffee grinder and sieved into several grades. Standard carotenoids (lycopene, -carotene and xanthophylls/lutein), HPLC grade of acetonitrile, 2-propanol and methanol used for analysis were purchased from Wako Pure Chemical Industries Ltd., Japan. CO2 was obtained from Uchimura Co., Japan. 2.2. SC-CO2 extraction Fig. 1 shows a schematic diagram of SC-CO2 extraction apparatus. The apparatus includes a chiller (Cooling Unit CLU-33, Iwaki Asahi Techno Glass, Japan), a pump for CO2 (Intelligent Prep. Pump. PU-2086 Plus, Jasco, Japan), a heating chamber (ST-110, ESPEC Corp., Japan), an extractor (Thar Tech, Inc., USA, 10 ml in volume), back pressure regulator (SCF-Bpg, Jasco, Japan), collection vials and a wet gas meter (Sinagawa Co., Japan). For process optimization, carotenoids were extracted under pressures of 150450 bar, temperatures of 4080 C and CO2 ow rates of 24 ml/min (based on the inlet pump ow rate) with 0.665 mm of particle size. In each experiment, approximately 3.5 g of samples were loaded into a 10 ml extraction vessel and the remaining volume was lled with glass beads in the bottom and top of the cell. The cell was placed in the heating chamber to maintain the operat-

ing temperature. The extract was collected in the vial at every 1040 min for 150 min, and weighed immediately after collection. 2.3. Chemical analysis Carotenoids composition was determined by highperformance liquid chromatography (HPLC) equipped with Intelligent UV/vis detector (UV-970, Jasco, Japan). Extract dissolved in solvent mixture consisting of 1:2 chloroform:acetone was injected through a 20 l loop and separated with a reversed-phase 5C18-MS Waters column (5 m; 4.6 mm 150 mm; Nacalai Tesque, Inc., Japan) at 30 C. The mobile phase was a quaternary solvent consisting of 39:52:5:4 acetonitrile:2-propanol:methanol:water owed at a ow rate of 1.4 ml/min. The separated carotenoids were monitored at 450 nm. The signals of the detector were recorded in BORWIN chromatography software. Peak identication of carotenoid prole was based on comparison of retention times and scanned spectra of lycopene, -carotene and lutein standards. Weight of lycopene, -carotene and lutein in the extract was calculated by comparison of the peak area and weight of lycopene, -carotene and lutein standard, respectively. 2.4. Statistical analysis According to the previous work [7], a full 33 factorial design was selected for optimization of operating condition of carotenoids extraction in SC-CO2 . The 27 experiments were performed and were run randomized. To determine possible interactions of process variables and their effect on the extraction yield of carotenoids and each carotene, an analysis of variance (ANOVA) was carried out according to the Excel Statistics 2004 program. The signicance level was stated at 95%, with p-value 0.05. 3. Result and discussion 3.1. Effect of extraction condition on carotenoids extracted Fig. 2 shows a chromatogram of carotenoids extracted at 450 bar, 60 C and 2 ml/min for 10 min. Based on the avail-

Fig. 1. Schematic diagram of supercritical CO2 extraction system.

Fig. 2. HPLC chromatogram of carotenoids extracted at 450 bar, 60 C and 2 ml/min for 10 min of extraction time.

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Fig. 3. Effect of pressure on (a) lycopene, (b) -carotene and (c) lutein extracted at 80 C and 4 ml/min. ( ) P = 150 bar; ( ) P = 300 bar; ( ) P = 450 bar.

able standard of carotenoids (lycopene, -carotene and luteion), carotenoids extracted mainly contain -carotene and lycopene. Small peak of lutein also can be detected in the extract. Hodisan et al. have identied that carotenoids from rosehip fruits contained cis- -carotene, -carotene, lycopene, rubixanthin, -cryptoxanthin and zeaxanthin mixed with lutein [2]. By comparison with Hodisan et al. [2] result, other peaks detected in the extract can be identied as zeaxanthin, cis-carotene, rubixanthin and -cryptoxanthin for peak nos. 2, 4, 6 and 7, respectively. In this work the four components could not be identied because the standards were not available. 3.1.1. Effect of pressure The effect of pressure on the amount of lycopene, -carotene and lutein extracted are shown in Fig. 3(a)(c), respectively. The amount of lycopene, -carotene and lutein extracted signicantly increased with increasing pressure. The dependency on the pressure was expected as the SC-CO2 density increases at higher pressure, and therefore the solvent power

to dissolve the carotenoids (lycopene, increases.

-carotene and lutein)

3.1.2. Effect of temperature Fig. 4(a)(c) shows the effect of temperature on the amount of lycopene, -carotene and lutein extracted, respectively. As shown in the gures, the amount of lycopene, extracted signicantly increased with increasing temperature, however the amount of -carotene and lutein extracted slightly increased with increasing temperature. Increasing temperature increases the solubility of the carotenoids, which results in higher yields. Instead of that the increasing temperature contributed to damage the particle cell walls, and as the result carotenoids availability for extraction was increased [8]. The effect of temperature on extraction was more difcult to assess than the effect of pressure. An increase in extraction temperature results in an increase in solute vapor pressure. The density of SC-CO2 at constant pressure, decreases as temperature increases, but the magnitude of such a density change becomes smaller at elevated pressures. This result is basically in agreement with those of other workers

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Fig. 4. Effect of temperature on (a) lycopene, (b) -carotene and (c) lutein extracted at 450 bar and 3 ml/min. ( ) T = 80 C; ( ) T = 60 C; ( ) T = 40 C.

[9,10] for lycopene and -carotene in the temperature range of 4080 C. 3.1.3. Effect of CO2 ow rate The effect of CO2 ow rate on the amount of lycopene, -carotene and lutein extracted are shown in Fig. 5(a)(c), respectively. As shown in the gures, generally the amount of lycopene, -carotene and lutein extracted signicantly increased with increasing CO2 ow rate. But for -carotene, extraction rate increased up to 3 ml/min of CO2 ow rate and becomes lower at higher CO2 ow rate. The increasing CO2 ow rate caused the increasing intermolecular interaction between CO2 and the carotenoids, thus increasing the solute dissolution. However the increasing CO2 ow rate also caused the shorter residence time to contact with -carotene; and as a result the decreasing carotene extracted. For this condition, the mass transfer was highly inuenced by the increasing CO2 ow rate and the intraparticle diffusion resistance was dominant. Based on the experimental result, the higher CO2 ow rate has improved the higher extraction efciency. Extraction process can be conducted at shorter time with higher extraction yield.

3.2. Process optimization and analysis of experimental design In process optimization, the effect of three main variables on SC-CO2 extraction was simultaneously studied using a threefactor design, with three levels for each factor (low (), medium (0) and high (+)). The selected factors were extraction temperature (T in C), SC-CO2 pressure (P in bar) and SC-CO2 ow rate (F in ml/min). The responses considered to evaluate the yield of carotenoids extraction. The extraction yield was obtained dividing weight of extract by weight of initial rosehip fruits sample. Table 1 lists the matrix for the experimental design, as well as the values given to each factor, which were selected according to the instrumental limitations, with the experimental results obtained. As shown in Table 1, maximum yield of total carotenoids was 20.88 mg/g feed obtained at experiment run #27 (80 C, 450 bar, 4 ml/min); maximum yield of lycopene was 14.37 mg/g feed found at experiment run #24 (80 C, 450 bar, 3 ml/min); and for -carotene and lutein were 1.017 mg/g feed and 16.84 g/g feed, respectively, found at experiment run #15 (60 C, 450 bar, 3 ml/min). As expected, higher recoveries were obtained at

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Fig. 5. Effect of CO2 ow rate on (a) lycopene, (b) -carotene and (c) lutein extracted at 300 bar and 40 C. ( ) F = 2 ml/min; ( ) F = 3 ml/min; ( ) F = 4 ml/min.

high pressure due to high density of CO2 . Higher temperature also exhibited higher recoveries due to higher solubility of carotenoids in SC-CO2 [11]. The results from the analysis of the experimental design for process optimization are shown in Table 2. The signicant effects and interaction between the range of variables studied and the analysis of variance of the extraction process are also given. The degree of signicance of each factor and interaction among variables are represented in the table by its p-value; when a factor and an interaction among variables have a p-value smaller than 0.05 it inuences the process in a signicant way for a condence level of 0.95 [7]. The results obtained show that the most inuential variable on the yield of total carotenoids extraction was temperature, with the statistical signicant effect p-value < 0.05. The other variables had a lesser inuence on the yield of total carotenoids extraction. For lycopene extraction, all variables were to be the inuential variables on the yield of lycopene extraction (p-value < 0.05). However, there is no possible interaction between temperature and pressure; temperature and ow rate; and pressure and ow rate on the yield of lycopene extraction (p-value > 0.05). For extraction of -carotene and lutein, the inuential variables were pressure and

CO2 ow rate without any interactions between temperature and pressure; temperature and ow rate; and pressure and ow rate. To determine the optimum condition in the range of variables process, yield of carotenoids extraction was correlated with temperature, pressure and CO2 ow rate based on the inuential variables and interaction among variables. The empirical correlation was obtained by tting the experimental data using solver in Excel 2003 program. The empirical correlations for the yield of total carotenoids, lycopene, -carotene and lutein are described in Eqs. (1)(4), respectively. Y is the yield of extract (mg/g feed and g/g feed for lutein), T is the temperature ( C), P is the pressure (bar) and F is the CO2 ow rate (ml/min). The resulting correlation coefcients are 0.870.91. Y = 0.261T + 0.004T 2 + 14.953 Y = 0.116T 0.001T 2 + 0.098P + 8.706F 1.202F 2 28.05 Y = 0.009P 0.060F + 0.022F 2 0.697 Y = 0.101P + 7.577F 0.925F 2 22.48 (2) (3) (4) (1)

S. Machmudah et al. / J. of Supercritical Fluids 44 (2008) 308314 Table 1 Factor levels and design matrix in the full 33 factorial design Factor Variable Temperature Pressure (bar) CO2 ow rate (ml/min) Run 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 T 0 0 0 0 0 0 0 0 0 + + + + + + + + + P 0 + 0 + 0 + 0 + 0 + 0 + 0 + 0 + 0 + F 0 0 0 + + + 0 0 0 + + + 0 0 0 + + + ( C) Key T P F Total carotenoids (mg/g feed) 10.35 10.72 13.30 12.43 14.82 16.10 18.02 14.20 11.21 11.58 17.10 12.29 13.95 15.41 19.75 15.46 15.74 14.36 17.48 16.15 15.08 15.59 16.17 16.93 19.65 20.42 20.88 Levels Low () 40 150 2 Lycopene (mg/g feed) 2.396 5.113 7.958 1.800 8.914 11.73 6.033 11.44 10.01 1.180 8.314 11.49 1.526 12.15 13.99 4.910 10.60 11.97 2.539 9.873 8.888 4.959 12.40 14.37 4.920 10.94 13.67 Middle (0) 60 300 3 -Carotene (mg/g feed) 0.398 0.573 0.764 0.304 0.934 0.972 0.726 0.951 0.805 0.168 0.740 0.873 0.154 0.943 1.017 0.579 0.815 0.822 0.233 0.781 0.704 0.445 0.794 0.950 0.359 0.769 0.841

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High (+) 80 450 4 Lutein ( g/g feed) 3.132 7.226 10.01 2.492 11.96 16.50 7.799 16.09 14.53 1.258 10.17 12.19 1.558 13.02 16.84 5.190 12.52 15.18 2.903 10.57 10.69 5.870 9.994 16.47 5.666 11.56 16.09

3.3. Extraction rate constant At constant volume, assuming that the mass transfer process could be formally treated as an irreversible rst-order reaction, the equation of extraction rate constant is [12]: k= 1 S0 ln t St (5)

Table 3 Extraction rate constants Pressure (bar) 150 300 450 450 450 450 Temperature ( C) 60 60 60 40 60 80 CO2 ow rate (ml/min) 3 3 3 4 4 4 k (min1 ) 3.590 103 5.268 103 5.851 103 3.344 103 4.503 103 5.638 103

where k is the extraction rate constant (min1 ); t the extraction time (min); S0 the total yield of extractible compounds (w/w) and St is the remained extractible compounds after
Table 2 Statistical signicant effects (p-value) for all variables and possible interactions among variables p-Value T Total carotenoids Lycopene -Carotene Lutein 0.012 0.040 0.588 0.954 P 0.778 0.000 0.000 0.000 F 0.064 0.002 0.047 0.002 TP 0.826 0.284 0.578 0.407 TF 0.372 0.527 0.746 0.525 PF 0.304 0.164 0.148 0.183

extraction time t (w/w). The extraction rate constant was calculated on the basis of obtained result of total carotenoids extraction yield. In this study S0 was 56.47 mg/g obtained via acetone soxhlet extraction for 5 h. Extraction rate constant k for each experimental point (extraction after 10, 20, 30, 50, 80, 110 and 150 min) was calculated and mean value is given in Table 3. Extraction time was conducted for 150 min because extraction rate was almost constant and approximately zero after 150 min of extraction. As expected, extraction rate constant k increased with increasing pressure and temperature.

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S. Machmudah et al. / J. of Supercritical Fluids 44 (2008) 308314 performance liquid chromatography, J. Pharm. Biomed. Anal. 16 (1997) 521528. M.D. Zlatanov, Lipid composition of Bulgarian chokeberry, black currant and rose hip seed oils, J. Sci. Food Agric. 79 (1999) 1620 1624. D.A. Daels-Rakotoarison, B. Gressier, F. Trotin, C. Brunet, M. Luyckx, T. Dine, F. Bailleul, M. Cazin, J.-C. Cazin, Effects of Rosa canina fruit extract on neutrophil respiratory burst, Phytother. Res. 16 (2002) 157161. T.W. Goodwin, Plant carotenoid research 19451985, J. Plant Physiol. 143 (1994) 440443. V. Illes, O. Szalai, M. Then, H. Daood, S. Perneczki, Extraction of hiprose fruit by supercritical CO2 and propane, J. Supercrit. Fluid 10 (1997) 209218. S. Machmudah, Y. Kawahito, M. Sasaki, M. Goto, Supercritical CO2 extraction of rosehip seed oil: fatty acids composition and process optimization, J. Supercrit. Fluid 41 (2007) 421428. S. Machmudah, A. Shotipruk, M. Goto, M. Sasaki, T. Hirose, Extraction of astaxanthin from Haematococcus pluvialis using supercritical CO2 and ethanol as entrainer, Ind. Eng. Chem. Res. 45 (2006) 36523657. E. Cadoni, M. Rita de Giorgi, E. Medda, G. Poma, Supercritical CO2 extraction of lycopene and -carotene from ripe tomatoes, Dyes Pigm. 44 (2000) 2732. E. Sabio, M. Lozano, V. Montero de Espinosa, R.L. Mendes, A.P. Pereira, A.F. Palavra, J.A. Coelho, Lycopene and -carotene extraction from tomato processing waste using supercritical CO2 , Ind. Eng. Chem. Res. 42 (2003) 66416646. K. Sasaki, Solubility of -carotene in dense carbondioxide from 308 to 323 and from 9.6 to 30 MPa, J. Chem. Eng. Data 37 (1992) 249251. W. Wang, J. Liu, W. He, D. Li, Kinetic study of ytterbium(III) extraction from sulfate medium with cyanex 923, J. Chem. Technol. Biotechnol. 82 (2007) 705710.

4. Conclusion Extraction process of carotenoids from rosehip fruit with SC-CO2 has been optimized using a full 33 factorial design. The analysis of experimental design and the optimum condition of the process were obtained. The optimum condition of lycopene extraction was found at 80 C, 450 bar, 3 ml/min; and for -carotene and lutein extraction was at 60 C, 450 bar, 3 ml/min. Extraction rate constant of total carotenoids extraction has been calculated based on the experimental results. The value of extraction rate constant k increased with increasing pressure and temperature. The values of extraction rate constant k were (3.3445.851) 103 min1 . Acknowledgment This work was partially supported by Kumamoto University 21st Century COE Program Pulsed Power Science. References
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