Phytochemical Extraction

Extraction of Phytochemicals Using Supercritical Carbon Dioxide Technique

Shanavas Bavu

Introduction
Phytochemicals such as phenolics, carotenoids, sterols, and alkaloids found in fruits, vegetables, spices and traditional herbal medicinal plants have demonstrated various health benefits, but the conventional technologies utilized to extract some of these phytochemicals have limitations. These methods, which use organic solvents, have disadvantages such as residues of solvent left in the final product, high processing temperatures, and emissions of volatile organic compounds. Today, one of the promising emerging extraction technologies that can overcome these disadvantages is supercritical fluid extraction (SCFE). This article discusses the SCFE technique using carbon dioxide (SC-CO2), the factors affecting extraction yield in SC-CO 2 extraction and various extraction parameters.
hytochemicals in fruits, vegetables, spices and traditional herbal medicinal plants have been found to play protective roles against many human chronic diseases including cancer and cardiovascular diseases (CVD). These diseases are associated with oxidative stresses caused by excess free radicals and other reactive oxygen species. Antioxidant phytochemicals exert their effect by neutralizing these highly reactive radicals. Among the tens of thousands of phytochemicals found in human diet or traditional medicines, polyphenols and carotenoids stand out as the two most important groups of natural antioxidants. Hundreds of carotenoids and thousands of polyphenols have been identified so far from various plants. A single plant could contain a highly complex profile of these compounds, which may exist at very low concentrations in the plant and is often susceptible to heat; air and light. This makes the isolation of these antioxidant phytochemicals a challenging proposition. Phytochemicals such as phenolics, carotenoids, sterols, and alkaloids are receiving increasing attention due to their demonstrated health benefits, but conventional technologies used to extract some of these phytochemicals may have limitations. Therefore, there is a need for emerging efficient technologies that are environmentally friendly, cost-effective, and can guarantee the sustainability of the food chain and product development. One of the most widely accepted emerging technologies is supercritical fluid extraction (SFE), which has already reached commercial application for many products, and is one of the most widely studied techniques.

Shanavas Bavu is Lead New Products division, Synthite Industries Ltd. His core experience is in process engineering and he has considerable exposure in setting up and commercializing of extraction technologies like SCFE and SCC. He has 5 years of experience in the oleoresin field and a total experience of 17 years in industry. Currently, he heads a team of researchers and application specialists in the New Products division at Synthite.

Sources of phytochemicals
Phenolic phytochemicals are mainly present in fruits, seeds, and herbs such as elderberry, grape, mate tea leaves, rose hip, Rosmarinus officinalis, Origanum dictamnus, Teucrium polium, and Styrax officinalis. The amount and type of phenolics and their conjugates differ markedly even in different tissues of the same species. For example, anthocyanins

82

Chemical Industry Digest. April 2012

CMYK

Phytochemical Extraction are the main polyphenols in the red grape skin, whereas flavan-3-ols are the major polyphenols in the grape seeds (Makris and others 2006). It has been proven that a diet rich in various classes of polyphenols (phenolic acids, flavonols, catechin monomers, proanthocyanidins, flavones, flavanones, anthocyanins) decreases the risk of premature mortality from major clinical conditions, including cancer and heart disease (Duthie and others 2003). Phenolic phytochemicals are known to exhibit several activities that are beneficial to health, such as antioxidant, anti-inflammatory, antihepatotoxic, antitumor, and antimicrobial activities (Middleton and others 2000; Rice-Evans and others 1996; Podsedek 2007; Kong and others 2003; Revilla and Ryan 2000). The consumption of polyphenols present in tea leaves, for example, can inhibit the formation and growth of tumors (Wang and others 1994; Inagake and others 1995; Hasegawa and others 1995). These effects are due to the properties of antioxidants to act as reducing agents by donating hydrogen, quenching singlet oxygen, acting as chelators, and trapping free radicals. a typical SCFE plant. Most SCFE processes use SC-CO2 because CO2 is nontoxic, non-flammable, and chemically inert and has a moderate and relatively easily achievable critical point of 31oC and 7.4 MPa. Some of the other advantages of using CO2 as the SCF are that CO2 is available in high purity at relatively low cost, it can be easily removed from the matrix after the SCFE process, and it can be easily separated from the extracted compounds. It is well documented that CO2, a nonpolar solvent, is best suited for the extraction of nonpolar organic compounds. Therefore, for the extraction of more polar compounds, the polarity of SC-CO2 can be increased by adding modifiers such as ethanol and water, which in turn increases the solubility of more polar compounds in the SCF. Other solvents such as nitrous oxide, ethane, and propane have been used as SCFs in various applications (Reid and others 1987). However, issues such as safety, disposal, toxic emissions, and flammability have limited their use. One important consideration for extraction with any solvent is the solubility of the phytochemical in the solvent. For example, when using supercritical fluid extraction, solubility is a strong function of SC-CO2 density and the properties of the solute such as molecular weight, polarity and vapour pressure. Phytochemicals are soluble in SC-CO2 to different extents depending on the temperature and pressure conditions. Solubility behavior of phenolics, carotenoids, sterols, and alkaloids in SC-CO2 has been previously reviewed (Choi et al 1998; Murga et al 2002, 2003; Gomez-Prieto et al 2002).

Extraction of phytochemicals from different sources

Various methods are used to extract phytochemicals from different natural sources. One of the widely used methods is supercritical fluid extraction (SCFE). A fluid is in its supercritical state when it is heated and compressed above its critical temperature (Tc) and critical pressure (Pc). Figure 1, shows the pressuretemperature (P-T) diagram with the supercritical fluid (SCF) region for carbon dioxide.

Other potential emerging methods to extract phytochemicals are PEF, MWE, UE, and OH. Emerging extraction methods have been studied for a number of agricultural In the supercritical state, the substance exists as a commodities (Saldana et al 2002a, b; Lianfu and Zelong single fluid phase with properties intermediate between 2008; Lopes and Bernardo-Gil 2005; Vatai et al 2009; those of liquids and gases: the densities are liquid like, Corrales and others 2008; Fincan and others 2004; whereas the diffusivities and viscosities are gas like Bernardo-Gil and others 2001; and many others). The ex(McHugh and Krukonis, 1994). Moreover, SCFs have zero traction efficiency of these techniques is affected by sevsurface tension, which allows easy penetration into most eral parameters such as particle size and moisture conmatrices including fruits and tent of the feed, extraction temvegetables. In addition, in the perature and pressure, power, supercritical state, SCFs are exA fluid is in its supercritical state solvent flow rate, extraction tremely sensitive to small time, frequency, and the use of when it is heated and compressed changes in temperature and presa co-solvent or a mixture of solabove its critical temperature (Tc) and sure such that a compound may vents. Therefore, the following critical pressure (Pc). Most SCFE probe extracted from a matrix at one discussion will focus on the imcesses use SC-CO 2 because CO 2 is set of conditions and then sepapact of these processing paramnontoxic, non-flammable, and chemirated from the SCF in a downeters on the yield of phytostream operation under a differcally inert and has a moderate and relachemicals obtained from fruits ent set of conditions. Figure 2 tively easily achievable critical point of and vegetables, nuts and seeds, shows the functional diagram of 31oC and 7.4 MPa. and herbs.

Chemical Industry Digest. April 2012

83

CMYK

Phytochemical Extraction

Factors affecting extraction yield in SC-CO2 extraction

Sample Preparation
Drying and grinding the samples are two important steps prior to extraction; because many fruits and vegetables contain 8095% moisture and grinding to achieve small particle size will favour the high contact surface area necessary for efficient extraction.

One important consideration for extraction with any solvent is the solubility of the phytochemical in the solvent. For example, when using supercritical fluid extraction, solubility is a strong function of SC-CO2 density and the properties of the solute such as molecular weight, polarity and vapour pressure.

(Vasapollo and others 2004).

Particle size
For the extraction of carotenoids from freeze-dried carrot using SC-CO2, the studies of Goto and others (1994) and Sun and Temelli (2006) show that a higher extraction yield was obtained with small carrot particles. In the study of Sun and Temelli (2006), the total carotenoid yield increased from 1,110 to 1,370 and 1,504 g/g dry carrots with particle sizes of 12 mm to 0.51 mm and 0.30.5 mm, respectively.

The amounts of -carotene extracted from carrots using SC-CO2 at 40oC and 50oC and 120327 bar for 8 hour extraction increased with temperature and pressure (Saldana and others 2006). Higher amounts of - carotene followed by -carotene and lutein were obtained. Furthermore, the quantities of - and -carotene extracted by SC-CO2 were one order of magnitude higher than those of lutein. This difference might facilitate the separation of lutein from - and -carotene by changing the operating conditions.

Flow rate and extraction time

Dynamic techniques for the extraction of carotenoids with SC-CO2 use flow rates that vary from 0.5 to 15 mL/ min (measured at extraction temperature and pressure) with different effects depending on the matrix (Rozzi and others 2002; Subra and others 1998; Saldana and others 2006). Subra and others (1998) extracted -carotene from 1 to 2.5 g freeze-dried carrots and studied the effect of flow rates (0.4 and 1.2 liter/min); they obtained higher yields of -carotene at a flow rate of 1.2 liter/min. Sun and Temelli (2006) also evaluated the effect of flow rate (0.5 and 1.0 liter/min) on the extraction of -carotene with SC-CO2 + canola oil. The total carotenoid yield increased with flow rate, ranging from 934.8 to 1,973.6 g/g dry 10,000

Moisture
Moisture had different effects on the extraction yield of phytochemicals. For example, the - and -carotene extraction yields using SC-CO2 increased from 184 to 599 g/g dry carrot and from 354 to 892 g/g, respectively, when moisture in the feed material was decreased from 84.6 to 0.8%. The lutein yield decreased from 55.3 to 13 g/g dry carrot with a decrease in moisture from 84.6 to 0.8% (Sun and Temelli 2006). For the extraction of lycopene from tomato with 5060% moisture content, only trace amounts of lycopene were reported (Vasapollo and others 2004).

1,000
pressure (bar)

Extraction parameters
Temperature and pressure
Most of the fruit and vegetables studies on extraction of phytochemicals using SC-CO2 were performed at a temperature range of 35110oC and a pressure range of 855 MPa. In general, extraction yield of carotenoids, such as - and -carotene, lutein, and lycopene, using SC-CO2 increased with temperature and pressure (Ollan-keto and others 2001; Gomez-Prieto and others 2003; Vasapollo and others 2004; Saldana and others 2006). For the extraction of lycopene at 60oC and 85oC, about 20% and 30% recovery was achieved in 80 min, respectively 84

100

10

1 200 250 300 temperature (K) Fig 1 Carbon dioxide pressuretemperature phase diagram adapted from McHugh and Krukonis 350 400

Chemical Industry Digest. April 2012

CMYK

Phytochemical Extraction CO2, to recover phytochemicals from a variety of biological plant sources. However, it is essential to study each plant composition individually because the pre-treatment of material and optimum extraction conditions will depend on the structure and on the composition of specific plant source.

PCV

CO2 make up Heater PCV Heater Cooler Separator 1

In addition, supercritical fluid technology allows a combination of extraction with fractionation to further separate bioactive components of interest. However, more research is needed to CO2 investigate the quality attributes of exstorage tracted phytochemicals, such as oxidative stability, chemical composition, Heater High pressure Cooler and stability of phytochemicals pump throughout extraction and storage by using emerging extraction technologies. Fig 2 Structure of a typical SCFE plant Finally, it is also necessary to better commucarrot at CO2 flow rates from 0.5 to 2 liter/min (measured nicate to consumers the advantages of these technologies at STP), respectively (Sun and Temelli, 2006). However, compared to conventional extraction technologies. the lycopene yield decreased from 38.8% to 8% as flow rate was increased from 2.5 to 15 mL/min (measured at extraction temperature and pressure) (Rozzi and others For many years, few new methods to extract 2002). phytochemicals were being developed. However, recently Use of co-solvent there has been a boom in emerging extraction technoloVarious co-solvents, such as acetone, ethanol, metha- gies. For a long time traditional methods using organic nol, hexane, dichloromethane, and water, have been used solvents were utilized to extract phytochemicals. These for the removal of carotenoids using SC-CO2 extraction methods presented disadvantages such as residues of sol(Ollanketo and others 2001). All these co-solvents except vent left in the final product, high processing temperawater (only 2% of recovery) increased the carotenoid re- tures, and emissions of volatile organic compounds. Tocovery. The use of vegetable oils such as hazelnut and day, one of the promising emerging extraction technolocanola oil as a co-solvent for the recovery of carotenoids gies that can overcome these disadvantages is SC-CO2 exfrom carrots and tomatoes have been reported (Sun and tractions. Extractor Separator 2

Conclusion

Temelli, 2006; Shi, 2001; Vasapollo and others 2004). When extracting without addition of co-solvent, the lycopene yield was below 10% for 2- to 5-hour extraction time, whereas in the presence of hazelnut oil, the lycopene yield increased to about 20% and 30% in 5 and 8 hour, respectively. The advantages of using vegetable oils as co-solvents are higher extraction yield; elimination of organic solvent addition, which needs to be removed later; and the enrichment of the oil with carotenoids that can be potentially used in a variety of product applications.

Future Trends
The literature reviewed in this article demonstrates the feasibility of using emerging technologies such as SC-

This method has the ability to recover phytochemicals such as phenolics, carotenoids, sterols, and alkaloids from a wide variety of agricultural plant sources. Extensive research within a large variety of plant materials such as fruits, vegetables, nuts and seeds, and herbs have shown that SC-CO2 is the most utilized emerging extraction method for the recovery of phytochemicals. There are many factors that affect extraction efficiency, such as sample preparation, moisture content, and the extraction parameters of temperature, pressure, solvent flow rate, extraction time, and use of a cosolvent. These parameters also have an impact on the various quality attributes such as color characteristics, flavor, and oxidative stability of the extracted phytochemicals and residual product.

Chemical Industry Digest. April 2012

85

CMYK