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Ultrasound-assisted extraction of capsaicinoids from Capsicum frutescens on a lab- and pilot-plant scale
Ultrasonics Sonochemistry 15 (2008) 1075–1079
Contents lists available at ScienceDirect
Ultrasonics Sonochemistry journal homepage: www.elsevier.com/locate/ultsonch
Ultrasound-assisted extraction of capsaicinoids from Capsicum frutescens on a lab- and pilot-plant scale Sumate Boonkird a, Chada Phisalaphong a, Muenduen Phisalaphong b,* a b
Research and Development Institute, The Government Pharmaceutical Organization, Bangkok 10400, Thailand Department of Chemical Engineering, Faculty of Engineering, Chulalongkorn University, Bangkok 10330, Thailand
a r t i c l e
i n f o
Article history: Received 5 February 2008 Received in revised form 2 April 2008 Accepted 22 April 2008 Available online 29 April 2008 Keywords: Extraction Ultrasonic effects Ultrasound-assisted extraction Capsaicinoids
a b s t r a c t The influence of operating parameters (solvent type, powder to solvent ratio and temperature) on the ultrasonically assisted extraction of capsaicinoids from dried Capsicum frutescens (fruit) was studied. From the economic perspective, the suitable condition for capsaicinoid extraction by indirect sonication in an ultrasonic bath with a working frequency of 35 kHz was at a ratio of 1 g of solid material: 5 ml of 95% (v/v) ethanol, 45 °C, where 85% of the capsaicinoids were removed from the raw material in 3 h. In an experimental pilot study in 20-l extraction tank at the fixed ultrasonic frequency of 26 kHz and 70 kHz, the recovery of capsaicinoids was 76% and 70%, respectively. It was shown that the ultrasonic extraction produced a significant reduction in extraction time at a lower operational temperature than under a conventional industrial hot maceration process. Ó 2008 Elsevier B.V. All rights reserved.
1. Introduction The chilli pepper is a popular plant found in many parts of the world. Chilli has been widely used as a food flavoring, a coloring agent, a feed additive in livestock and in food and pharmaceutical industries. Capsaicin and its analogues, called capsaicinoids are pungent compounds of Capsicum fruits. Capsaicin, the major pungent compound, is an amide derivative of vanillylamine and 8methlnon-trans-6-enoic acid. Its hot flavor for about 95% of the pungency is caused by three major substances, capsaicin, dihydrocapsaicin and nordihydrocapsacin of capsaicinoids [1]. Capsaicinoids are known for their pharmacological properties for instance as chemoprotectors against mutagenesis or tumorigenesis [2], as antimicrobials [3], as antioxidants [4], for their anticancer effect [5], their analgesic effects [6], and their effect on the neuronal responsible for pain transmission and neurogenic inflammation [7]. The classical techniques for capsainoid extraction are maceration [8], magnetic stirring [9] and Soxhlet [10]. Ultrasonics has been proven to assist the solvent extraction of bioactive compounds from herbs [11–15]. The application of ultrasound-assisted extraction (UAE) offers many advantages including the reduction of solvents, temperature and the time for extraction, which is very useful for the extraction of thermolabile and in unstable compounds. The ultrasonic enhancement of supercritical
* Corresponding author. Tel.: +662 218 6875; fax: +662 218 6877. E-mail address: [email protected] (M. Phisalaphong). 1350-4177/$ - see front matter Ó 2008 Elsevier B.V. All rights reserved. doi:10.1016/j.ultsonch.2008.04.010
extraction of pungent compounds from ginger owing to physical effects on the surface of particles was reported [16]. Recently, new techniques such as microwave-assisted extraction [17] and supercritical CO2 extraction [18] have been applied to the extraction of capsaicinoids from chilli powder. However, relative to those techniques, the use of UAE is more convenient and affordable. In addition, UAE can be simply industrially employed in local companies. Owing to its cavitational phenomenon, the UAE can be applied in place of the boiling maceration method. While many papers have been published dealing with the UAE of different plant materials [11–16,19–21], only a preparative method of capsaicinoids for HPLC analysis by using ultrasonic extraction with acetone, methanol and acetonitrile as solvents has been reported [19]. In order to improve capsaicinoids extraction efficiency by UAE, the influence of operating parameters on a lab- and pilot-plant scale were examined in this study. The obtained results were compared with conventional extraction methods, maceration and Soxhlet.
2. Materials and method 2.1. Plant materials Fruits of Capsicum frutescens were purchased from a local market in Mae Chan district, Chiangrai, Thailand in the form of dried chilies. For UAE lab study, the dried chilli pepper was grounded with a brabender (No. 1855334, type 880805, Germany) to obtain 3 mm particle size. For UAE pilot study, the sample size of dried
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chilli pepper was reduced to 13 mm by using Hammer mill Model H-15 Hosokawa Micron Corp. (Osaka, Japan). The ground samples were packed in plastic bags purged with nitrogen, and stored at 4 °C before use.
in a small aluminum tray was placed in a vacuum oven at a pressure of 90 mbar and 105 °C for 4 h and then weighed.
2.2. Soxhlet extraction
3.1. Effect of ultrasonication and its extraction time
A classical Soxhlet apparatus was employed in which 25 g of the sample was placed into the cartridge with 200 ml of 95%v/v ethanol in 250 ml flask. Extraction at boiling point (78.1 °C) was carried out for 5 h.
Ground dried chilli peppers were added with a ratio of powder (g): 95% (v/v) ethanol (ml) of 1:8, in 250 ml Erlenmeyer flasks and ultrasonicated in an ultrasonic cleaning bath with a working frequency of 35 kHz at 45 °C for a period of 3 h. Extraction by maceration at 45 °C was used as the control system. The effect of ultrasonication and its extraction time on the recovery of capsaicinoids is shown in Fig. 1. The extraction rate of capsaicinoids was very high during the first 5 min of the extraction. After that, the recovery of capsaicinoids gradually increased with the extraction time. Capsaicinoid recovery by UAE for 3 h was then compared to that of maceration (15 h) and Soxhlet (5 h) (Table 1). The percentage recovery of capsaicinoids by maceration, Soxhlet and UAE was 79.4, 92.0 and 87.4, respectively. UAE gave 10% greater capsaicinoid recovery compared with maceration but an approximately 5% lower yield compared with Soxlet extraction. The Soxhlet extraction gave the highest percentage recovery because the extraction was performed at boiling point temperature (78.1 °C) and the extraction was done all the time with fresh solvent, unloaded with substances, having therefore a greater concentration gradient as driving force, comparing with all other extractions. UAE significantly improved the extraction yield. Since ultrasound could accelerate swelling and hydration and caused an enlargement in the pores of the plant cell walls, it resulted in a better mass transfer of solute constituents from the plant materials to solvent. The disruption of plant cells by microjet after the cavitation bubble collapsed could increase the rate of solvent penetration into plant tissue [20,21]. The release rate of capsaicinoids was very
2.3. Maceration The experiment was performed in a 250 ml flask filled with 25 g of ground sample in 200 ml of 95% v/v ethanol. The extraction was carried out at 45 °C, 250 rpm for 15 h. 2.4. UAE lab study The experiment was carried out in triplicate using an ultrasonic cleaning bath with a working frequency of 35 kHz and the power of 600 W (Bandelin Sonorex Super RK 1050, Germany). The bath was a rectangular container (50 cm 60 cm 20 cm) connected to a temperature-controlled bath (Polyscience 9610, USA). A set of 250 ml Erlenmeyer flasks filled with 25 g of ground samples of 3 mm particle size and assigned solvent was immersed into the ultrasonic bath under a controlled water level at about 10 mm from the bottom of the bath. The milling degree of the sample material played an important role in the extraction process; however, the reducing sizes of the material particles less than 3 mm particle size caused inconvenience in the filtration step. 2.5. UAE pilot study
3. Results and discussion
The experiment was carried out in a 20-l extraction tank consisting of transducers bonded to walls with double output of ultrasonic frequencies (26 and 70 kHz) and ultrasound electric power of 1.08 kW (Jinning Sinobes Electronic, China). The stirrer speed was 16 rpm from an explosion proof motor. Three kilograms of course ground dried chilli pepper was employed for each batch with the selected conditions from the previous UAE lab study. The milling method for material preparation in the pilot study followed the conventional milling method of the industrial scale hot maceration. The sample size of dried chilli pepper was reduced to 13 mm particle size by using Hammer mill. The particle size was larger than that in the lab study in order to ease the filtration step. 2.6. Analytical method for capsaicinoids The concentrations of capsaicinoids were determined following the method of the Association of Analytical Communities (AOAC) method 995.03 using a high-performance liquid chromatography (HPLC) system; Thermo Separation Product (TSP) consisting of the Spectra System P1000 solvent delivery and UV-6000LP diode array UV visible detector (Thermo Separation Product, USA). A column (150 4.6 mm ID) was packed with C18 reverse phase of 5 lm particle size. Isocratic 40% acetonitrile in 1% acetic acid (v/ v) was used as a mobile phase with a flow rate of 1.5 ml/min. The injection volume was 20 ll and the UV wavelength was 280 nm. The concentration of capsaicinoids was a combination of capsaicin, dihydrocapsaicin and nordihydrocapsacin concentrations. Measured retention times and peak areas represented at least triplicate injections. The capsaicinoids concentration was quantified on the basis of a corresponding calibration curve using pure substances. For dry weight determination, 2 g of the sample
Fig. 1. Effect of ultrasonication and its extraction time on the extraction of capsaicinoids at 45 °C in 95% (v/v) ethanol: (j) UAE and () maceration.
S. Boonkird et al. / Ultrasonics Sonochemistry 15 (2008) 1075–1079
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Table 1 Capsaicinoid recovery by UAE compared to that of maceration and Soxhlet Methods
Time (h)
Temperature (°C)
Powder:solvent (g/ml)
Recovery (%)
Maceration Soxhlet UAE UAE UAE
15 5 3 3 3
45.0 78.1 45.0 45.0 45.0
1:8 1:8 1: 8 1:6 1:5
79.4 92.0 87.4 84.5 84.9
high at the beginning due to the effect of the capsaicinoid concentration gradient between the solvent and the plant material and the ease of extraction from the outer part of particles in the early period. Afterwards the release rate of capsaicinoids considerably decreased because of the lower concentration gradient and because the remaining capsaicinoids were located in the inner part. 3.2. Effect of extraction temperature The effect of temperature on the release of capsaicinoids extracted by 95% ethanol and acetone as a solvent is shown in Figs. 2 and 3, respectively. It was found that with 95% ethanol as a solvent, the increase in temperature from 30 °C to 45 °C enhanced the capsaicinoid recovery. The percentage recovery leveled out when the temperature was raised from 45 °C to 60 °C. Temperature affected many physical properties such as viscosity, diffusivity, solubility, vapor pressure and surface tension. However, the main effect of ultrasounds was cavitational bubbles collapse. The observed effects appeared to be due to acoustic cavitation, which was supported by the fact that a higher efficiency was observed at 45 °C rather than 60 °C, whereas the physical agitation generated by US was not responsible. At 45 °C, the number of cavitation bubbles increase and their collapse is powerful enough to act on extraction. At 60 °C, although the number of bubbles is higher, their collapse is less efficient. Therefore, the cavitational effects at 45 °C exhibited a stronger intensity than at 60 °C. Higher vapor pressure, especially near the boiling point of the liquid reduced the cavitation to nearly zero. It has been previously explained that at
Fig. 3. Effect of extraction temperature on the UAE of capsaicinoids in acetone: () 45 °C and (j) 30 °C.
high vapor pressure, more bubbles were created but they collapsed with lower intensity owing to the smaller internal/external pressure difference [14]. Using acetone as a solvent, no significant enhancement in extraction efficiency was observed when the temperature was raised from 30 °C to 45 °C (Fig. 3). Ethanol has usually been considered as a less efficient solvent compared with acetone due to its higher viscosity and lower diffusivity. However, under ultrasounds, acetone is less effective than alcohol. In this study, UAE yield relatively increased when 75–95% (v/v) ethanol was used as a solvent compared with acetone. Since the vapor pressure of acetone was much higher than ethanol and water, therefore more bubbles but inefficient collapse were obtained, especially at the employed temperatures. Consequently, the enhancement of extraction efficiency by the cavitation effect using acetone as a solvent was lower compared to ethanol or water. A similar observation of greater increase in extraction efficiency as a result of UAE in ethanol compared with acetone was reported previously [14]. 3.3. Effect of ratio of water in ethanol
Fig. 2. Effect of extraction temperature on the UAE of capsaicinoids in 95% (v/v) ethanol: () 60 °C, (j) 45 °C and (N) 30 °C.
The ratio of water in the ethanol solution had a significant effect on the extraction of capsaicinoids (Fig. 4). For all controlled temperatures, it was found that 50% ethanol was not effective for capsaicinoid extraction. This should be due to the differences in polarity between the solvent and the extracted compounds. The polarity of capsaicinoids is much lower than water, so that it is not well solubilized in 50% (v/v) ethanol. At 45 °C, capsaicinoid recovery was slightly greater using 95% ethanol compared with 70% ethanol, while, at 60 °C, 75% ethanol was found to be the more effective solvent. Water content in the applied solvent played an important role in the extraction. Swelling of the plant material by water enhanced the extraction efficiency. In addition, ultrasound facilitated the hydration processes of dried materials [11]. Ultrasound enlarged the pores of the cell walls so that the diffusion process and mass transfer were improved [12]. The intensity of ultrasonic cavitation in the ethanol mixture in the presence of water was also increased as a result of the increase in surface ten-
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100
Recovery (%)
80
60
40
20
0
30
45
60
Temperature (ºC) Fig. 4. Effect of ratio of water in the ethanol solution on the UAE of capsaicinoids at 30–60 °C: ( ) 50% ethanol, (j) 75% ethanol and (h) 95% ethanol.
sion and the decrease in viscosity. However, from the economic perspective, it was costly to use 75% (v/v) ethanol as a solvent since the removal of the solvent at the final step required high energy consumption. Therefore, 95% (v/v) ethanol was chosen as the applied solvent for the further study.
Fig. 5. Recovery of capsaicinoids on the UAE pilot study at 26 kHz and 70 kHz in 95% (v/v) ethanol at 45 °C: () 26 kHz and (j) 70 kHz.
3.4. Effect of solvent to material ratio The extraction efficiency at different ratios of chilli powder (g) to 95% ethanol (ml) is shown in Table 1. It was found that when the ratio was raised from 1:5 to 1:8, the recovery only slightly increased. However, the result could differ if a large amount of solvent was employed [19]. On the other hand, using a large amount of solvent was not considered cost-effective due to the high operating cost of solvents and energy consumption. Consequently, the ratio of the dry weight of chilli powder to 95% (v/v) ethanol was selected at 1:5. 3.5. UAE pilot study An experimental pilot study was carried out in a 20-l extraction tank consisting of a double output of ultrasonic frequencies of 26 and 70 kHz. From the previous lab study, the selected conditions for the UAE pilot study were at a controlled temperature of 45 °C, with a ratio of 1 g of ground dried chilli pepper per 5 ml of 95% (v/v) ethanol. The particle size of ground pepper (13 mm) used in the pilot study was larger than that in the lab study (3 mm) in order to ease the filtration step. Nonetheless, the sonication could itself serve to mill the sample material producing tiny plant’s particles, which were hard to filter. The study for the effect of ultrasonic frequency was performed by maintaining a constant ultrasound electric power at 1.08 kW as the frequency increased from 26 kHz to 70 kHz. The experimental result is shown in Fig. 5. It was found that the percentage recovery of capsaicinoids at 26 kHz (76.4%) was higher than that at 70 kHz (69.6%). A decrease in the amount and intensity of cavitation in liquids was obtained at high frequency [22]. At high frequency, the rarefaction (and compression) cycles time for bubbles to grow to a size sufficient to cause disruption of the liquid was shorter. Sound intensity is proportional to the square of the product of frequency and amplitude [23], therefore with a constant intensity, higher magnitudes value was obtained at lower frequency. Consequently, better cavitation effect at 26 kHz in comparison to that at 70 kHz due to higher amplitude resulted in higher recovery of capsaicinoid. The similar effect of sonic frequency on metal extraction was previously reported [23].
The lower percentage recovery from the pilot scale extraction compared with the lab scale could be according to the variation in particle size, the operating frequency, the electric power and differences in the geometry and mixing intensity between the two systems. A particle size was the key parameter in the extraction process. The capsaicinoid extraction by the hot maceration process on an industrial scale (1500-L) was performed with the optimized procedure for comparison. Chilli powder with the same particle size (13 mm) as that for the pilot UAE was soaked overnight in 95% (v/v) ethanol at 30 °C. The solution was then heated at boiling point (78.1 °C) for 3 h. After that the extract was drawn to the bottom of the extractor. The solid residues were separated by the filtration unit. The obtained solution was mixed thoroughly and a 15 ml of sample was drawn for HPLC analysis. The recovery of capsaicinoids extracted by the hot maceration process was 81.8%, which was about 7.0% higher than that from the UAE pilot scale. The procedure implied a good swelling and also a high temperature extraction that usually leaded to a better wash of the particles during extraction. Although, the yield of capsaicinoids from the UAE pilot scale was slightly lower than that from industrial scale hot maceration, the result showed that the potential use of UAE was promising for extraction on an industrial scale. UAE could shorten the extraction time and lower the operating temperature, which resulted in considerably lower operating costs. Moreover, from the chromatogram observation, there were lower impurity peaks in the HPLC chromatogram of the extract obtained by the UAE method (data not shown), which could be due to the reduction in extraction time and temperature. This was certainly one of the greater advantages of UAE.
4. Conclusion Ultrasound-assisted extraction of capsaicinoids from C. frutescens (fruit) on a lab- and pilot-plant scale was investigated. The rate of capsaicinoid release was very fast during the first 5 min of the extraction and significantly decreased after that. The increased
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temperature from 30 °C up to 45 °C enhanced the extraction of capsaicinoids by 95% (v/v) ethanol, however, the percentage recovery leveled out when the temperature was raised from 45 °C to 60 °C. UAE yield relatively increased when 75–95% (v/v) ethanol was use as a solvent compared with acetone. On the basis of economic efficiency, the suitable procedure for extracting capsaicinoids from chilli powder was at the ratio of 1 g dried powder per 5 ml of 95% ethanol with a controlled temperature of 45 °C for a period of 3 h. During the UAE pilot study at constant ultrasound electric power of 1.08 kW, the operation at 26 kHz gave a higher percentage recovery of capsaicinoids than that obtained at 70 kHz. The percentage recovery of capsaicinoids from the pilot scale UAE was slightly lower than that obtained from industrial scale hot maceration. Overall, ultrasound proved to assist solvent extraction both on a small and large scale since it produced a significant reduction in temperature and the time required to extract capsaicinoids from their sources and a high quality product was expected. Acknowledgement The authors acknowledge equipment support from Research and Development Institute, The Government Pharmaceutical Organization. References [1] A.M. Krajewska, J.J. Powers, Gas chromatography of methyl derivatives of naturally occurring capsaicinoids, J. Chromatogr. 409 (1987) 223–233. [2] Y.-J. Surh, R.C.-J. Lee, K.-K. Park, S.T. Mayne, A. Liem, J.A. Miller, Chemoprotective effects of capsaicin and diallyl sulfide against mutagenesis or tumorigenesis by vinyl carbamate and N-nitrosodimethylamine, Carcinogenesis 16 (1995) 2467–2471. [3] R.H. Cichewicz, The antimicrobial properties of Chile peppers (Capsicum species) and their uses in Mayan medicine, J. Ethnopharmacol. 52 (1996) 61–70. [4] D.E. Hendersen, A.M. Slickman, Quantitative HPLC determination of the antioxidant activity of capsaicin on the formation of liquid hydroperoxides of linoleic acid: a comparative study against BHT and melatonin, J. Agric. Food Chem. 47 (1999) 2563–2570. [5] Y.J. Surh, More than spice: capsaicin in hot chili peppers makes tumor cells commit suicide, J. Natl. Cancer Inst. 94 (2002) 1263–1265.
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[6] E. Kaale, A. Van Schepdael, E. Roets, J. Hoogmartens, Determination of capsaicinoids in topical cream by liquid-liquid extraction and liquid chromatography, J. Pharm. Biomed. Anal. 30 (2002) 1331–1337. [7] J. Szolcsanyi, Forty years in capsaicin research for sensory pharmacology and physiology, Neuropeptides 38 (2004) 377–384. [8] P. Kirschbaum-Titze, C. Hiepler, E. Mueller-Seitz, M. Petz, Pungency in paprika (Capsicum annuum). 1. Decrease of capsaicinoid content following cellular disruption, J. Agric. Food Chem. 50 (2002) 1260–1263. [9] M. Contreras-Padilla, E.M. Yahia, Changes in capsaicinoids during development, maturation, and senescence of Chile peppers and relation with peroxidase activity, J. Agric. Food Chem. 46 (1998) 2075–2079. [10] F. Korel, N. Bagdatlioglu, M.Ö. Balaban, Y. Hisßil, Ground red peppers: capsaicinoids content, scoville scores, and discrimination by an electronic nose, J. Agric. Food Chem. 50 (2002) 3257–3261. [11] M. Vinatoru, An overview of the ultrasonically assisted extraction of bioactive principles from herbs, Ultrason. Sonochem. 8 (2001) 303–313. [12] M.I. Soares Melecchi, V.F. Pe´res, C. Dariva, C.A. Zini, F.C. Abad, M.M. Martinez, E.B. Caramão, Optimization of the sonication extraction method of Hibiscus tiliaceus L. flowers, Ultrason. Sonochem. 13 (2006) 242–250. [13] M. Sališvová, Š. Toma, T.J. Mason, Comparison of conventional and ultrasonically assisted extractions of pharmaceutically active compounds from Salvia officinalis, Ultrason. Sonochem. 4 (1997) 131–134. [14] S. Hemwimol, P. Pavasan, A. Shotipruk, Ultrasound-assisted extraction of anthraquinones from roots of Morinda citrifolia, Ultrason. Sonochem. 13 (2006) 543–548. [15] D.T. Velicˇkovic´, D.M. Milenovic´, M.S. Ristic´, Kinetics of ultrasonic extraction of extractive substances from garden (Salvia officinalis L.) and glutinous (Salvia glutinosa L.) sage, Ultrason. Sonochem. 13 (2006) 150–156. [16] S. Balachandran, S.E. Kentish, R. Mawson, M. Ashokkumar, Ultrasonic enhancement of the supercritical extraction from ginger, Ultrason. Sonchem. 13 (2006) 471–479. [17] F.B. Gerardo, M. Palma, C.G. Barroso, Determination of capsaicinoids in peppers by microwave-assisted extraction-high-performance liquid chromatography with fluorescence detection, Anal. Chim. Acta 578 (2006) 227–233. [18] A. Perva-Uzunalic´, M. Škerget, B. Weinreich, Zˇ. Knez, Extraction of chilli pepper (var. Byedige) with supercritical CO2: effect of pressure and temperature on capsaicinoid and colour extraction efficiency, Food Chem. 87 (2004) 51–58. [19] R. Karnka, M. Rayanakorn, S. Watanesk, Y. Vaneesorn, Optimization of highperformance liquid chromatographic parameters for the determination of capsaicinoid compounds using the simplex method, Anal. Sci. 18 (2002) 661– 665. [20] L. Paniwnyk, E. Beaufoy, J.P. Lorimer, The extraction of rutin from flower buds of Sophora japonica, Ultrason. Sonochem. 8 (2001) 299–301. [21] M. Toma, M. Vinatoru, L. Paniwnyk, T.J. Mason, Investigation of the effects of ultrasound on vegetal tissues during solvent extraction, Ultrason. Sonochem. 8 (2001) 137–142. [22] T.J. Mason, J.P. Lormier, Sonochemistry Theory Application and Uses of Ultrasound in Chemistry, John Wiley & Sons, New York, 1988. [23] K.M. Swamy, K.L. Narayana, Intensification of leaching process by dualfrequency ultrasound, Ultrason. Sonochem. 8 (2001) 341–346.
Contents lists available at ScienceDirect
Ultrasonics Sonochemistry journal homepage: www.elsevier.com/locate/ultsonch
Ultrasound-assisted extraction of capsaicinoids from Capsicum frutescens on a lab- and pilot-plant scale Sumate Boonkird a, Chada Phisalaphong a, Muenduen Phisalaphong b,* a b
Research and Development Institute, The Government Pharmaceutical Organization, Bangkok 10400, Thailand Department of Chemical Engineering, Faculty of Engineering, Chulalongkorn University, Bangkok 10330, Thailand
a r t i c l e
i n f o
Article history: Received 5 February 2008 Received in revised form 2 April 2008 Accepted 22 April 2008 Available online 29 April 2008 Keywords: Extraction Ultrasonic effects Ultrasound-assisted extraction Capsaicinoids
a b s t r a c t The influence of operating parameters (solvent type, powder to solvent ratio and temperature) on the ultrasonically assisted extraction of capsaicinoids from dried Capsicum frutescens (fruit) was studied. From the economic perspective, the suitable condition for capsaicinoid extraction by indirect sonication in an ultrasonic bath with a working frequency of 35 kHz was at a ratio of 1 g of solid material: 5 ml of 95% (v/v) ethanol, 45 °C, where 85% of the capsaicinoids were removed from the raw material in 3 h. In an experimental pilot study in 20-l extraction tank at the fixed ultrasonic frequency of 26 kHz and 70 kHz, the recovery of capsaicinoids was 76% and 70%, respectively. It was shown that the ultrasonic extraction produced a significant reduction in extraction time at a lower operational temperature than under a conventional industrial hot maceration process. Ó 2008 Elsevier B.V. All rights reserved.
1. Introduction The chilli pepper is a popular plant found in many parts of the world. Chilli has been widely used as a food flavoring, a coloring agent, a feed additive in livestock and in food and pharmaceutical industries. Capsaicin and its analogues, called capsaicinoids are pungent compounds of Capsicum fruits. Capsaicin, the major pungent compound, is an amide derivative of vanillylamine and 8methlnon-trans-6-enoic acid. Its hot flavor for about 95% of the pungency is caused by three major substances, capsaicin, dihydrocapsaicin and nordihydrocapsacin of capsaicinoids [1]. Capsaicinoids are known for their pharmacological properties for instance as chemoprotectors against mutagenesis or tumorigenesis [2], as antimicrobials [3], as antioxidants [4], for their anticancer effect [5], their analgesic effects [6], and their effect on the neuronal responsible for pain transmission and neurogenic inflammation [7]. The classical techniques for capsainoid extraction are maceration [8], magnetic stirring [9] and Soxhlet [10]. Ultrasonics has been proven to assist the solvent extraction of bioactive compounds from herbs [11–15]. The application of ultrasound-assisted extraction (UAE) offers many advantages including the reduction of solvents, temperature and the time for extraction, which is very useful for the extraction of thermolabile and in unstable compounds. The ultrasonic enhancement of supercritical
* Corresponding author. Tel.: +662 218 6875; fax: +662 218 6877. E-mail address: [email protected] (M. Phisalaphong). 1350-4177/$ - see front matter Ó 2008 Elsevier B.V. All rights reserved. doi:10.1016/j.ultsonch.2008.04.010
extraction of pungent compounds from ginger owing to physical effects on the surface of particles was reported [16]. Recently, new techniques such as microwave-assisted extraction [17] and supercritical CO2 extraction [18] have been applied to the extraction of capsaicinoids from chilli powder. However, relative to those techniques, the use of UAE is more convenient and affordable. In addition, UAE can be simply industrially employed in local companies. Owing to its cavitational phenomenon, the UAE can be applied in place of the boiling maceration method. While many papers have been published dealing with the UAE of different plant materials [11–16,19–21], only a preparative method of capsaicinoids for HPLC analysis by using ultrasonic extraction with acetone, methanol and acetonitrile as solvents has been reported [19]. In order to improve capsaicinoids extraction efficiency by UAE, the influence of operating parameters on a lab- and pilot-plant scale were examined in this study. The obtained results were compared with conventional extraction methods, maceration and Soxhlet.
2. Materials and method 2.1. Plant materials Fruits of Capsicum frutescens were purchased from a local market in Mae Chan district, Chiangrai, Thailand in the form of dried chilies. For UAE lab study, the dried chilli pepper was grounded with a brabender (No. 1855334, type 880805, Germany) to obtain 3 mm particle size. For UAE pilot study, the sample size of dried
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S. Boonkird et al. / Ultrasonics Sonochemistry 15 (2008) 1075–1079
chilli pepper was reduced to 13 mm by using Hammer mill Model H-15 Hosokawa Micron Corp. (Osaka, Japan). The ground samples were packed in plastic bags purged with nitrogen, and stored at 4 °C before use.
in a small aluminum tray was placed in a vacuum oven at a pressure of 90 mbar and 105 °C for 4 h and then weighed.
2.2. Soxhlet extraction
3.1. Effect of ultrasonication and its extraction time
A classical Soxhlet apparatus was employed in which 25 g of the sample was placed into the cartridge with 200 ml of 95%v/v ethanol in 250 ml flask. Extraction at boiling point (78.1 °C) was carried out for 5 h.
Ground dried chilli peppers were added with a ratio of powder (g): 95% (v/v) ethanol (ml) of 1:8, in 250 ml Erlenmeyer flasks and ultrasonicated in an ultrasonic cleaning bath with a working frequency of 35 kHz at 45 °C for a period of 3 h. Extraction by maceration at 45 °C was used as the control system. The effect of ultrasonication and its extraction time on the recovery of capsaicinoids is shown in Fig. 1. The extraction rate of capsaicinoids was very high during the first 5 min of the extraction. After that, the recovery of capsaicinoids gradually increased with the extraction time. Capsaicinoid recovery by UAE for 3 h was then compared to that of maceration (15 h) and Soxhlet (5 h) (Table 1). The percentage recovery of capsaicinoids by maceration, Soxhlet and UAE was 79.4, 92.0 and 87.4, respectively. UAE gave 10% greater capsaicinoid recovery compared with maceration but an approximately 5% lower yield compared with Soxlet extraction. The Soxhlet extraction gave the highest percentage recovery because the extraction was performed at boiling point temperature (78.1 °C) and the extraction was done all the time with fresh solvent, unloaded with substances, having therefore a greater concentration gradient as driving force, comparing with all other extractions. UAE significantly improved the extraction yield. Since ultrasound could accelerate swelling and hydration and caused an enlargement in the pores of the plant cell walls, it resulted in a better mass transfer of solute constituents from the plant materials to solvent. The disruption of plant cells by microjet after the cavitation bubble collapsed could increase the rate of solvent penetration into plant tissue [20,21]. The release rate of capsaicinoids was very
2.3. Maceration The experiment was performed in a 250 ml flask filled with 25 g of ground sample in 200 ml of 95% v/v ethanol. The extraction was carried out at 45 °C, 250 rpm for 15 h. 2.4. UAE lab study The experiment was carried out in triplicate using an ultrasonic cleaning bath with a working frequency of 35 kHz and the power of 600 W (Bandelin Sonorex Super RK 1050, Germany). The bath was a rectangular container (50 cm 60 cm 20 cm) connected to a temperature-controlled bath (Polyscience 9610, USA). A set of 250 ml Erlenmeyer flasks filled with 25 g of ground samples of 3 mm particle size and assigned solvent was immersed into the ultrasonic bath under a controlled water level at about 10 mm from the bottom of the bath. The milling degree of the sample material played an important role in the extraction process; however, the reducing sizes of the material particles less than 3 mm particle size caused inconvenience in the filtration step. 2.5. UAE pilot study
3. Results and discussion
The experiment was carried out in a 20-l extraction tank consisting of transducers bonded to walls with double output of ultrasonic frequencies (26 and 70 kHz) and ultrasound electric power of 1.08 kW (Jinning Sinobes Electronic, China). The stirrer speed was 16 rpm from an explosion proof motor. Three kilograms of course ground dried chilli pepper was employed for each batch with the selected conditions from the previous UAE lab study. The milling method for material preparation in the pilot study followed the conventional milling method of the industrial scale hot maceration. The sample size of dried chilli pepper was reduced to 13 mm particle size by using Hammer mill. The particle size was larger than that in the lab study in order to ease the filtration step. 2.6. Analytical method for capsaicinoids The concentrations of capsaicinoids were determined following the method of the Association of Analytical Communities (AOAC) method 995.03 using a high-performance liquid chromatography (HPLC) system; Thermo Separation Product (TSP) consisting of the Spectra System P1000 solvent delivery and UV-6000LP diode array UV visible detector (Thermo Separation Product, USA). A column (150 4.6 mm ID) was packed with C18 reverse phase of 5 lm particle size. Isocratic 40% acetonitrile in 1% acetic acid (v/ v) was used as a mobile phase with a flow rate of 1.5 ml/min. The injection volume was 20 ll and the UV wavelength was 280 nm. The concentration of capsaicinoids was a combination of capsaicin, dihydrocapsaicin and nordihydrocapsacin concentrations. Measured retention times and peak areas represented at least triplicate injections. The capsaicinoids concentration was quantified on the basis of a corresponding calibration curve using pure substances. For dry weight determination, 2 g of the sample
Fig. 1. Effect of ultrasonication and its extraction time on the extraction of capsaicinoids at 45 °C in 95% (v/v) ethanol: (j) UAE and () maceration.
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Table 1 Capsaicinoid recovery by UAE compared to that of maceration and Soxhlet Methods
Time (h)
Temperature (°C)
Powder:solvent (g/ml)
Recovery (%)
Maceration Soxhlet UAE UAE UAE
15 5 3 3 3
45.0 78.1 45.0 45.0 45.0
1:8 1:8 1: 8 1:6 1:5
79.4 92.0 87.4 84.5 84.9
high at the beginning due to the effect of the capsaicinoid concentration gradient between the solvent and the plant material and the ease of extraction from the outer part of particles in the early period. Afterwards the release rate of capsaicinoids considerably decreased because of the lower concentration gradient and because the remaining capsaicinoids were located in the inner part. 3.2. Effect of extraction temperature The effect of temperature on the release of capsaicinoids extracted by 95% ethanol and acetone as a solvent is shown in Figs. 2 and 3, respectively. It was found that with 95% ethanol as a solvent, the increase in temperature from 30 °C to 45 °C enhanced the capsaicinoid recovery. The percentage recovery leveled out when the temperature was raised from 45 °C to 60 °C. Temperature affected many physical properties such as viscosity, diffusivity, solubility, vapor pressure and surface tension. However, the main effect of ultrasounds was cavitational bubbles collapse. The observed effects appeared to be due to acoustic cavitation, which was supported by the fact that a higher efficiency was observed at 45 °C rather than 60 °C, whereas the physical agitation generated by US was not responsible. At 45 °C, the number of cavitation bubbles increase and their collapse is powerful enough to act on extraction. At 60 °C, although the number of bubbles is higher, their collapse is less efficient. Therefore, the cavitational effects at 45 °C exhibited a stronger intensity than at 60 °C. Higher vapor pressure, especially near the boiling point of the liquid reduced the cavitation to nearly zero. It has been previously explained that at
Fig. 3. Effect of extraction temperature on the UAE of capsaicinoids in acetone: () 45 °C and (j) 30 °C.
high vapor pressure, more bubbles were created but they collapsed with lower intensity owing to the smaller internal/external pressure difference [14]. Using acetone as a solvent, no significant enhancement in extraction efficiency was observed when the temperature was raised from 30 °C to 45 °C (Fig. 3). Ethanol has usually been considered as a less efficient solvent compared with acetone due to its higher viscosity and lower diffusivity. However, under ultrasounds, acetone is less effective than alcohol. In this study, UAE yield relatively increased when 75–95% (v/v) ethanol was used as a solvent compared with acetone. Since the vapor pressure of acetone was much higher than ethanol and water, therefore more bubbles but inefficient collapse were obtained, especially at the employed temperatures. Consequently, the enhancement of extraction efficiency by the cavitation effect using acetone as a solvent was lower compared to ethanol or water. A similar observation of greater increase in extraction efficiency as a result of UAE in ethanol compared with acetone was reported previously [14]. 3.3. Effect of ratio of water in ethanol
Fig. 2. Effect of extraction temperature on the UAE of capsaicinoids in 95% (v/v) ethanol: () 60 °C, (j) 45 °C and (N) 30 °C.
The ratio of water in the ethanol solution had a significant effect on the extraction of capsaicinoids (Fig. 4). For all controlled temperatures, it was found that 50% ethanol was not effective for capsaicinoid extraction. This should be due to the differences in polarity between the solvent and the extracted compounds. The polarity of capsaicinoids is much lower than water, so that it is not well solubilized in 50% (v/v) ethanol. At 45 °C, capsaicinoid recovery was slightly greater using 95% ethanol compared with 70% ethanol, while, at 60 °C, 75% ethanol was found to be the more effective solvent. Water content in the applied solvent played an important role in the extraction. Swelling of the plant material by water enhanced the extraction efficiency. In addition, ultrasound facilitated the hydration processes of dried materials [11]. Ultrasound enlarged the pores of the cell walls so that the diffusion process and mass transfer were improved [12]. The intensity of ultrasonic cavitation in the ethanol mixture in the presence of water was also increased as a result of the increase in surface ten-
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100
Recovery (%)
80
60
40
20
0
30
45
60
Temperature (ºC) Fig. 4. Effect of ratio of water in the ethanol solution on the UAE of capsaicinoids at 30–60 °C: ( ) 50% ethanol, (j) 75% ethanol and (h) 95% ethanol.
sion and the decrease in viscosity. However, from the economic perspective, it was costly to use 75% (v/v) ethanol as a solvent since the removal of the solvent at the final step required high energy consumption. Therefore, 95% (v/v) ethanol was chosen as the applied solvent for the further study.
Fig. 5. Recovery of capsaicinoids on the UAE pilot study at 26 kHz and 70 kHz in 95% (v/v) ethanol at 45 °C: () 26 kHz and (j) 70 kHz.
3.4. Effect of solvent to material ratio The extraction efficiency at different ratios of chilli powder (g) to 95% ethanol (ml) is shown in Table 1. It was found that when the ratio was raised from 1:5 to 1:8, the recovery only slightly increased. However, the result could differ if a large amount of solvent was employed [19]. On the other hand, using a large amount of solvent was not considered cost-effective due to the high operating cost of solvents and energy consumption. Consequently, the ratio of the dry weight of chilli powder to 95% (v/v) ethanol was selected at 1:5. 3.5. UAE pilot study An experimental pilot study was carried out in a 20-l extraction tank consisting of a double output of ultrasonic frequencies of 26 and 70 kHz. From the previous lab study, the selected conditions for the UAE pilot study were at a controlled temperature of 45 °C, with a ratio of 1 g of ground dried chilli pepper per 5 ml of 95% (v/v) ethanol. The particle size of ground pepper (13 mm) used in the pilot study was larger than that in the lab study (3 mm) in order to ease the filtration step. Nonetheless, the sonication could itself serve to mill the sample material producing tiny plant’s particles, which were hard to filter. The study for the effect of ultrasonic frequency was performed by maintaining a constant ultrasound electric power at 1.08 kW as the frequency increased from 26 kHz to 70 kHz. The experimental result is shown in Fig. 5. It was found that the percentage recovery of capsaicinoids at 26 kHz (76.4%) was higher than that at 70 kHz (69.6%). A decrease in the amount and intensity of cavitation in liquids was obtained at high frequency [22]. At high frequency, the rarefaction (and compression) cycles time for bubbles to grow to a size sufficient to cause disruption of the liquid was shorter. Sound intensity is proportional to the square of the product of frequency and amplitude [23], therefore with a constant intensity, higher magnitudes value was obtained at lower frequency. Consequently, better cavitation effect at 26 kHz in comparison to that at 70 kHz due to higher amplitude resulted in higher recovery of capsaicinoid. The similar effect of sonic frequency on metal extraction was previously reported [23].
The lower percentage recovery from the pilot scale extraction compared with the lab scale could be according to the variation in particle size, the operating frequency, the electric power and differences in the geometry and mixing intensity between the two systems. A particle size was the key parameter in the extraction process. The capsaicinoid extraction by the hot maceration process on an industrial scale (1500-L) was performed with the optimized procedure for comparison. Chilli powder with the same particle size (13 mm) as that for the pilot UAE was soaked overnight in 95% (v/v) ethanol at 30 °C. The solution was then heated at boiling point (78.1 °C) for 3 h. After that the extract was drawn to the bottom of the extractor. The solid residues were separated by the filtration unit. The obtained solution was mixed thoroughly and a 15 ml of sample was drawn for HPLC analysis. The recovery of capsaicinoids extracted by the hot maceration process was 81.8%, which was about 7.0% higher than that from the UAE pilot scale. The procedure implied a good swelling and also a high temperature extraction that usually leaded to a better wash of the particles during extraction. Although, the yield of capsaicinoids from the UAE pilot scale was slightly lower than that from industrial scale hot maceration, the result showed that the potential use of UAE was promising for extraction on an industrial scale. UAE could shorten the extraction time and lower the operating temperature, which resulted in considerably lower operating costs. Moreover, from the chromatogram observation, there were lower impurity peaks in the HPLC chromatogram of the extract obtained by the UAE method (data not shown), which could be due to the reduction in extraction time and temperature. This was certainly one of the greater advantages of UAE.
4. Conclusion Ultrasound-assisted extraction of capsaicinoids from C. frutescens (fruit) on a lab- and pilot-plant scale was investigated. The rate of capsaicinoid release was very fast during the first 5 min of the extraction and significantly decreased after that. The increased
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temperature from 30 °C up to 45 °C enhanced the extraction of capsaicinoids by 95% (v/v) ethanol, however, the percentage recovery leveled out when the temperature was raised from 45 °C to 60 °C. UAE yield relatively increased when 75–95% (v/v) ethanol was use as a solvent compared with acetone. On the basis of economic efficiency, the suitable procedure for extracting capsaicinoids from chilli powder was at the ratio of 1 g dried powder per 5 ml of 95% ethanol with a controlled temperature of 45 °C for a period of 3 h. During the UAE pilot study at constant ultrasound electric power of 1.08 kW, the operation at 26 kHz gave a higher percentage recovery of capsaicinoids than that obtained at 70 kHz. The percentage recovery of capsaicinoids from the pilot scale UAE was slightly lower than that obtained from industrial scale hot maceration. Overall, ultrasound proved to assist solvent extraction both on a small and large scale since it produced a significant reduction in temperature and the time required to extract capsaicinoids from their sources and a high quality product was expected. Acknowledgement The authors acknowledge equipment support from Research and Development Institute, The Government Pharmaceutical Organization. References [1] A.M. Krajewska, J.J. Powers, Gas chromatography of methyl derivatives of naturally occurring capsaicinoids, J. Chromatogr. 409 (1987) 223–233. [2] Y.-J. Surh, R.C.-J. Lee, K.-K. Park, S.T. Mayne, A. Liem, J.A. Miller, Chemoprotective effects of capsaicin and diallyl sulfide against mutagenesis or tumorigenesis by vinyl carbamate and N-nitrosodimethylamine, Carcinogenesis 16 (1995) 2467–2471. [3] R.H. Cichewicz, The antimicrobial properties of Chile peppers (Capsicum species) and their uses in Mayan medicine, J. Ethnopharmacol. 52 (1996) 61–70. [4] D.E. Hendersen, A.M. Slickman, Quantitative HPLC determination of the antioxidant activity of capsaicin on the formation of liquid hydroperoxides of linoleic acid: a comparative study against BHT and melatonin, J. Agric. Food Chem. 47 (1999) 2563–2570. [5] Y.J. Surh, More than spice: capsaicin in hot chili peppers makes tumor cells commit suicide, J. Natl. Cancer Inst. 94 (2002) 1263–1265.
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