Review Received: 2 December 2013

Revised: 1 July 2014

Accepted article published: 22 July 2014

Published online in Wiley Online Library:

(wileyonlinelibrary.com) DOI 10.1002/jsfa.6833

Phytosterols and their extraction from various plant matrices using supercritical carbon dioxide: a review Md. Salim Uddin,a,b Md. Zaidul Islam Sarker,a* Sahena Ferdosh,c Md. Jahurul Haque Akanda,d Mst. Sabina Easmin,a Siti Hadijah Bt Shamsudina and Kamaruzzaman Bin Yunuse Abstract Phytosterols provide important health benefits: in particular, the lowering of cholesterol. From environmental and commercial points of view, the most appropriate technique has been searched for extracting phytosterols from plant matrices. As a green technology, supercritical fluid extraction (SFE) using carbon dioxide (CO2 ) is widely used to extract bioactive compounds from different plant matrices. Several studies have been performed to extract phytosterols using supercritical CO2 (SC-CO2 ) and this technology has clearly offered potential advantages over conventional extraction methods. However, the efficiency of SFE technology fully relies on the processing parameters, chemistry of interest compounds, nature of the plant matrices and expertise of handling. This review covers SFE technology with particular reference to phytosterol extraction using SC-CO2 . Moreover, the chemistry of phytosterols, properties of supercritical fluids (SFs) and the applied experimental designs have been discussed for better understanding of phytosterol solubility in SC-CO2 . © 2014 Society of Chemical Industry Keywords: bioactive compounds; phytosterols; supercritical carbon dioxide extraction; benefit of phytosterols

INTRODUCTION A broad range of bioactive compounds such as phytochemicals, pharmaceutics, flavors, fragrances and pigments originate from plants. Natural bioactive compounds have diversified structures and functionalities that provide excellent molecular properties for the production of nutraceuticals, functional foods and food additives.1 Over the last few decades, natural bioactive compounds with potential for the treatment and prevention of human diseases have attracted much attention in many laboratories and industries. Bioactive compounds are used as natural product-derived therapeutic agents or as disease-preventing nutrients.2 Therefore, the growing market in functional foods and nutraceuticals is targeting all types of bioactive compounds, including lipids, carotenoids, phenolic compounds and plant sterols, for their health benefits. Plant sterols, known generally as phytosterols, are essential components of the membrane lipid bilayer3,4 and are found in all plant tissues and especially in seeds, vegetables and cereals.5 They perform functions in plants similar to those of cholesterol in animals and as precursors of important biomolecules such as sex hormones and vitamins.6 In particular, phytosterols have received much attention owing to their capability to lower serum cholesterol levels in humans,7,8 resulting in significant reductions in the risk of heart disease. Moreover, they show anti-inflammatory, antibacterial, anti-ulcerative and antitumor properties,9 and therefore make a great contribution to the value of natural products as medicinally active nutraceuticals. Conventional methods for lipid extraction from natural sources involve cooking, pressing and liquid extraction. The most J Sci Food Agric (2014)

common conventional process for extraction is liquid solvent extraction using chloroform, hexane, toluene, petroleum ether, acetone etc.10 Extraction and isolation from several natural sources using liquid solvent produce large quantity of waste organic solvents that are harmful to human health as well as the environment.11 Moreover, thermolabile compounds may be degraded in conventional separation methods due to applying high temperature for processing or evaporating organic solvents. Nowadays, people are concerned by the health, environmental and safety hazards associated with the use of organic solvents in food and pharmaceutical



Correspondence to: Md Zaidul Islam Sarker, Department of Pharmaceutical Technology, Kulliyyah of Pharmacy, International Islamic University Malaysia, Kuantan Campus, 25200 Kuantan, Pahang, Malaysia. E-mail: [email protected]

a Kulliyyah of Pharmacy, International Islamic University Malaysia, Kuantan Campus, 25200 Kuantan, Pahang, Malaysia b Department of Biochemistry and Molecular Biology, University of Rajshahi, Rajshahi 6205, Bangladesh c School of Industrial Technology, Universiti Sains Malaysia, 11800 Minden, Penang, Malaysia d Department of Food Science and Nutrition, Faculty of Applied Sciences, UCSI University, 56000 Kuala Lumpur, Malaysia e Kulliyyah of Science, International Islamic University Malaysia, Kuantan Campus, 25200, Kuantan, Pahang, Malaysia

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www.soci.org industries and the possible solvent contamination of the final products. Therefore, convenient separation methods and technologies have been researched to promote product quality and the quantity of active natural products without creating environmental problems.12 Supercritical fluid extraction (SFE) is an efficient alternative to the use of organic solvents for the extraction of functional and nutraceutical components from natural sources.11,13,14 Among the supercritical fluids (SFs), carbon dioxide (CO2 ) is widely used because supercritical CO2 (Sc-CO2 ) provides some advantages over conventional extraction processes.15,16 Several research works have been carried out on phytosterol extraction and fractionation using SC-CO2 from various plant sources.12,17 – 22 These studies showed that SFE is an effective technology for the recovery of phytosterol. Conventional and non-conventional extraction methods for bioactive compounds have been extensively reviewed elsewhere.23 – 25 The aim of this review is to consolidate and give detailed and updated information on phytosterol extraction using SC-CO2 and on the application of phytosterols and to provide some insight into future potential.

PHYTOSTEROLS: CLASSIFICATION AND BIOSYNTHESIS Phytosterols represent a diverse group of natural compounds belonging to the triterpene group, which are important structural components of plant membranes. They comprise 28 or 29 carbon-containing alcohols and are similar to cholesterol in vertebrates, both in structure (four-ring steroid nucleus, a 3𝛽-hydroxyl group and often a 5,6 double bond) and function (stabilization of phospholipid bilayers in cell membranes). Phytosterols consist of an additional methyl or ethyl group and/or double bond and most of them have a nine- to ten-carbon side chain instead of the eight-carbon side chain of cholesterol (Fig. 1).26,27 Plant sterols are classified in a variety of ways based on the presence or absence of a methyl group in different positions. According to the number of methyl groups at the C4 position (Fig. 1), sterols can be categorized into three subclasses: (i) 4,4-dimethylsterols; (ii) 4-monomethylsterols; (iii) 4-desmethylsterols.28 Usually, plants are able to demethylate sterols at the C4 position, but probably unable to carry out the reverse order.29,30 The 4,4-dimethylsterols are early plant sterol precursors, while the intermediate plant sterols are 4-monomethylsterols. The 4-desmethysterols are the final precursor of the most common phytosterols. Therefore, phytosterols are to be considered as 4-desmethylsterols of the cholestane series, all of which have double bonds at the C5 position of the ring.31,32 According to alkylation at the C24 position in the side chain, sterols can be classified as 24-desmethylsterols (without an alkyl group), 24-methylsterols and 24-ethylsterols. These sterols are also denoted as C8, C9 and C10 sterols, respectively, depending on the number of carbons in the sterol side chain. Additionally, 24-desmethylsterols, 24-methylsterols and 24-ethylsterols are sometimes referred as C27, C28 and C29 sterols, respectively, depending on the total number of carbons in the sterol molecule.30 Naturally, sterols are found as free sterols or conjugate forms, but they are most commonly present in free form anchored in the plasma membrane of all tissues. There are a few conjugates in which the 3𝛽-hydroxyl group of free sterol is esterified to a fatty acid or a hydroxycinnamic acid, or glycosylated with a hexose sugar (usually glucose). Steryl glycosides can also be esterified with a fatty acid to form acetylated steryl glycosides. Among the conjugates, the esterified sterols with fatty acids are the

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most abundant in plants having biological importance owing to their connection with the storage of sterols.30,32,33 The inbred constituents of cereals – specifically corn, wheat, rye and rice and fruit and vegetables are phytostanols, which comprise a fully saturated subgroup of phytosterols. The concentrations of these phytostanols are usually lower than those of unsaturated phytosterols.34,35 Several research studies have been carried out to elucidate the biosynthetic pathway of phytosterols.36 – 39 It is still complicated to gather detailed knowledge regarding phytosterol biosynthesis from individual literature studies. However, terpenes are derived from the isoprene units. Isoprenoids, the very early precursor of phytosterol, are derived by two pathways: the plastidial 2C-methyl-D-erythritol-4-phosphate (MEP) pathway40 or the mevalonate (MVA) pathway, which is localized to the cytosol, endoplasmic reticulum and peroxisomes.37,41 Recent evidence suggests that phytosterols are usually synthesized via the MVA pathway.37,42,43 Phytosterols biosynthesis can be separated in two stages: the MVA pathway and enzymatic steps specific to sterols.43 In the first stage, squalene, a 30-carbon linear isoprenoid intermediate, is synthesized from acetyl-CoA via mevalonate and then epoxidized to 2,3-oxidosqualene. These steps are similar for the biosynthesis of animal sterols. The differences in the biosynthesis of sterols between higher plants and animals are generally recognized from the cyclization step of 2,3-oxidosqualene.44 The initial step specific to phytosterol synthesis is the cyclization of 2,3-oxidosqualene to form cycloartenol, which is the first precursor of phytosterols.30,43 This triterpene molecule is methylated at the C24 position. Finally, sterol molecules are synthesized by further modification with a series of oxidation, reduction and demethylation reactions. Figure 2 shows the biosynthesis of the main phytosterols via the MVA pathway. More than 200 types of phytosterols have been reported.26 The most common phytosterols found in nature are 𝛽-sitosterol, campesterol and stigmasterol. 𝛽-Sitosterol and stigmasterol belong to the 24-ethylsterol group, whereas campesterol is a 24-methysterol.

HEALTH BENEFITS OF PHYTOSTEROLS Phytosterols have important roles in many areas such as in pharmaceuticals (production of therapeutic steroids), nutrition and cosmetics. They are commonly used as a value-added additive in food and cosmetic industries because of their health benefits.45 It has long been known that phytosterols as a dietary component have a tremendous health benefit in reducing serum cholesterol level as well as the risk of cardiovascular disease in humans.46 – 50 Several studies have reported that appropriate consumption of phytosterols could reduce the risk of heart disease by up to 40%, depending upon age and other dietary factors.48,51 Hypercholesterolemic persons have used 𝛽-sitosterol as a supplement and drug for lowering serum cholesterol level since 1950.52 Several clinical studies also suggest that phytosterols minimize the plasma level of total cholesterol and low-density-lipoprotein (LDL) cholesterol by reducing intestinal cholesterol absorption.46,47,53,54 Until now, the mechanism of action of phytosterols has not been precisely known, but there are several theories regarding the lowering of cholesterol level in blood plasma.32,48 One theory proposed that the marginally soluble cholesterol in intestine is precipitated into a non-absorbable state by the presence of added phytosterols. Another is that, for absorption into the bloodstream through intestinal cells, cholesterol has to enter mixed micelles consisting

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(a)

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(b) 242 241 21

1 19 2

HO

34

11

12 9

10

20

22

18 17

13 8

14

24 23

16

26 25

27

15

7

5 6

29

HO

28

(c)

HO Figure 1. (a) Phytosterol structure with carbon numbering according to IUPAC-IUB, 1989; (b) 𝛽-sitosterol; (c) cholesterol.

of bile salts and phospholipids. In these micelles, cholesterol is marginally soluble and is displaced by phytosterols, resulting in the prevention of cholesterol absorption. Recently, Brauner et al.55 reported that phytosterols reduce cholesterol absorption by inhibition of 27-hydroxycholesterol generation. An in vitro test has been performed using 𝛽-sitosterol and campesterol with Caco-2 and HepG2 cells. Micelles were prepared by mixing these cells with appropriate volumes of taurocholate, cholesterol and oleic acid, or taurocholate and phosphatidylcholine, followed by loading with normal and radiolabeled phytosterols. Net uptake was measured by counting the radioactivity in cells and media. Phytosterols have also other health-promoting effects including anticancer activity, atherosclerotic activity, anti-inflammatory activity and antioxidative activity. Several studies found that phytosterols show toxicity to breast and colon cancer cells.56 – 59 Gregg60 reported that phytosterols may be one of the active ingredients in saw palmetto, which has anticancer activity against prostate cancer cells. Epidemiological studies also suggest that a diet rich in phytosterol reduces common cancers such as lung, stomach, colon, breast and prostate cancer.50 Some researchers have provided evidence that phytosterols, especially Δ5 -avenasterol, have antioxidant activity and also antipolymerization properties.61 – 63 An in vitro study has been performed by Ferretti et al.64 based on the peroxidation of LDL. Phytosterols in ethanol were mixed with LDL isolated from blood plasma and suspended in phosphate buffer. The suspension was incubated for different times at 37 ∘ C in the presence of copper sulfate. The extent of lipid peroxidation of LDL was evaluated by measuring the level of conjugated diene formation and monitoring the increase in optical density. Authors found that phytosterols protect LDL from peroxidation. Recently, Burg et al.65 reported that dietary intake of phytosterol blends mainly containing stigmasterol might be beneficial in preventing Alzheimer’s disease (AD). In respect to AD, it was found that J Sci Food Agric (2014)

phytosterols are not as amyloidogenic as cholesterol; in particular, stigmasterol reduces amyloidogenic processing and therefore might be beneficial in AD. There is also a negative impact of phytosterols on the human body. Excess intake of phytosterols may reduce the blood levels of carotenoids as well as vitamin A, which are essential for the visual function of the eye.66 SFs A supercritical fluid (SF) defines the state of a substance in which the temperature and pressure are raised above their critical values. SFs possess the properties of both a gas and liquid and hence they exist as a fluid instead of either a gas or a liquid. The properties of SFs have been discussed in detail elsewhere.67 – 69 The properties of SFs can be explained based on the density, diffusivity and viscosity. A phase diagram of pressure and temperature can illustrate the physical stage of a substance. In the pressure–temperature phase diagram, sublimation, melting and boiling lines demarcate the regions of gas, liquid and solid state, respectively (Fig. 3). The vapor pressure begins at the triple point and ends at the critical point. The critical region emerges at the critical point. The critical region shows only one phase, which possesses a few properties of both gas and liquid.67 Solvating strength varies with the density of SFs. Generally, higher density of solvents that depends on temperature and pressure provides greater efficiency of extraction. Therefore, density is taken into account first regarding the solvating power of SFs.70 The density of SFs is similar to liquid, whereas the viscosity and diffusivity lie between gas-like and liquid-like values. Therefore, they can diffuse easily through solids like a gas and dissolve materials like a liquid. This property of SFs has gained much attention for their applicability in different fields, especially extraction of bioactive compounds from natural sources. Extraction is also possible by solvents under subcritical conditions, though the efficiency is low in most extractions. Subcritical fluids refer to fluids with pressure and temperature below the critical point.

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Acetyl-CoA

MdS Uddin et al.

Mevalonate Squalene

o

2,3-oxidosqualene

HO

Cycloartenol

HO

24-methylenelophenol

HO

Campesterol

HO

HO

Stigmasterol

β−Sitosterol

Figure 2. Biosynthesis of the main phytosterols via the MVA pathway.

SFs have some key advantages as compared to liquid solvents: (i) the solvating power of SFs depends on density, which can be easily controlled by the modification of pressure and temperature; (ii) SFs have higher diffusion coefficient and lower viscosity than liquid solvents, which greatly favors mass transfer.25,71 SFE Technology In the past two decades, SFE technology has been applied extensively to extract bioactive and valuable compounds from various matrices at laboratory and commercial scales. Despite some drawbacks at the commercial scale, SFE technology has been considered as a promising technology for the food and pharmaceutical industries. The ultimate goal of SFE is high selectivity, short extraction time and high purity of product, while being non-hazardous

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to health as well as the environment.25 CO2 is the first choice as a supercritical solvent for the extraction of bioactive compounds, because it is odorless, colorless, non-flammable, non-toxic, inert to most materials, safe, inexpensive and recyclable, and can be used under mild operational conditions (a critical temperature of 31.1 ∘ C and a critical pressure of 7.38 MPa).15,71 A great benefit provided by the SF separation process using CO2 as a solvent is that the solvent is separated from the products by only a depressurizing process. Moreover, due to low critical temperature, SC-CO2 extraction can be performed at a moderate temperature that prevents the degradation of thermally sensitive compounds.72,73 Therefore, CO2 as a supercritical solvent has been used in more than 90% of SF extraction of compounds from natural sources.71 CO2 is non-polar and hence the low polarity nature of CO2 limits its use

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Phytosterols and their extraction using supercritical carbon dioxide

Supercritical fluid Critical point Critical pressure

Pressure

Solid state

Liquid state

Subcritical region Gaseous state

Triple point

Temperature

Critical temperature

Figure 3. Pressure–temperature phase diagram of a substance.

for the extraction of polar bioactive compounds. This limitation can be successfully overcome by the use of small amount of polar co-solvent with SC-CO2 . Addition of co-solvents to SC-CO2 significantly increases the solubility of polar compounds.25 Ethanol has been mostly used as co-solvent because of its low boiling point and its availability in food-compatible grade. Co-solvent may influence the SC-CO2 extraction of polar compounds in three ways, depending on the sample type as well as on the analyte–sample matrix affinity. First, co-solvent produces an analyte–co-solvent interaction that increases the solubility of the analytes in SC-CO2 ; second, polar co-solvents can interact with the sample matrix and hence facilitate the desorption of analytes easily; and third, swelling of the sample matrix with co-solvent favors the penetration of SC-CO2 into the matrix, which increases the extraction of polar compounds. A schematic diagram of a simple SFE process is shown in Fig. 4. Generally, this apparatus is equipped with an extraction vessel, high-pressure pump for fluid pumping, co-solvent pump, chiller, back-pressure regulator, separator and gas flow meter. In addition, temperature is controlled by various devices such as a water bath, oven or electric heater. CO2 was pumped into the extraction vessel by high-pressure pump up to the desired pressure. The back-pressure regulator is used to control the pressure of CO2 . A co-solvent pump is attached to a SFE unit to pump polar solvents, enhancing the extraction of polar compounds. Flow rates and accumulated gas volume passing through the apparatus were measured using a gas flow meter. The extracts were collected using a separating vessel by depressurizing CO2 . Depending on the sample particle size, some filler materials are used in the extraction vessel to obtain maximum efficiency of the SFE process. Fine particles can usually speed up the extraction process, but at the same time may interfere in maintaining a proper flow rate of CO2 . Therefore, some rigid and inert materials such as sea sand and glass beads/crystal balls are packed with the ground materials to maintain the desired permissibility of the particle bed during the extraction process.25,74

SC-CO2 EXTRACTION OF PHYTOSTEROLS Even though SC-CO2 extraction is an effective and environmentally friendly technique for the separation of phytosterols from various plant sources, limited studies have been performed specific to phytosterol extraction. In the SFE process, first priority should be given to optimizing the extraction conditions. The optimum values J Sci Food Agric (2014)

www.soci.org for different variables that influence the SFE increase remarkably the yield of the compound of interest.75 For an efficient extraction process, all extraction parameters such as pressure, temperature, particle size, moisture content of the sample, flow rate of CO2 , extraction time and solvent-to-feed ratio are optimized.76 A variety of sample matrices and their pretreatment and storage conditions also affect the yield of extract and target compounds. However, the impact of extraction variables on the yield of phytosterol has been partially evaluated only in a few studies. Table 1 shows the operating conditions for phytosterol extraction from various plant matrices using SC-CO2 . Generally, the amount of extract yield and compound of interest has been affected by changing of pressure and temperature. The pressure–temperature effect on the yield can be explained in a variety of ways. The solvent density decreases with increasing temperature and hence reduces the solubility of compounds by reducing the solvating power of SC-CO2 . In contrast, the vapor pressure of the compound increases due to increasing temperature leading to increasing the yield of the extract by enhancing the solubility of compounds in SC-CO2 . In addition, during extraction mass transfer kinetics are enhanced due to increase in diffusivity with temperature. On the other hand, the solvent density increases with increase of pressure and thereby increases the solubility of the compound of interest. High pressure should not be applied for all extraction purposes because it increases repulsive solute–solvent interaction, which necessitates complex extraction and analysis processes of the compound of interest.77 Since phytosterols are a component of membrane lipid, breakdown of the cell membrane may speed up the extraction by increasing exposure of the components to SFs and thus may decrease the operating cost. Phytosterols in extracts are mainly analyzed by high-performance liquid chromatography (HPLC), gas chromatography–mass spectrometry (GC-MS), ultra-performance liquid chromatography–mass spectrometry (UPLC-MS) and gas chromatography–flame ionization detection (GC-FID). Sajfrtová et al.12 performed extraction of 𝛽-sitosterol from sea buckthorn (Hippophae rhamnoides L.) seeds at a pressure ranging from 15 to 60 MPa and a temperature ranging from 40 to 80 ∘ C. The CO2 flow rate was 0.8 g min−1 and the total extraction time was 9 h. To maximize the concentration of 𝛽-sitosterol in the extract, the optimum pressure and temperature were 60 MPa and 40 ∘ C with respect to solvent consumption and extraction rate (Fig. 5). Usually, the yield of the extracts increased with pressure and solvent consumed in a certain period of time. The phytosterol yield from buckthorn seed also increased with the increase in pressure. There is a complex effect of pressure and temperature on the phytosterol yield obtained by SC-CO2 extraction. At high temperature, the phytosterol yield was lower since the density of the solvent was dominant over vapor pressure of the solute. In another study, the same extraction conditions were applied to extract phytosterol from H. rhamnoides seeds.78 The optimum pressure and temperature for 𝛽-sitosterol extraction were 28 MPa and 60 ∘ C for fresh sample, and 60 MPa and 60 ∘ C for stored sample. The yield of 𝛽-sitosterol from storage seed was lower than that of fresh seed. It can be stated that 𝛽-sitosterol may interact strongly with the matrices and/or can be probably be lost due to storage of the seed. From these studies it was also found that extraction of 𝛽-sitosterol was completed earlier than the total yield of extract. Phytosterol-enriched oil extraction using SC-CO2 from Kalahari melon seed has been carried out by Nyam et al.79 Authors considered the extraction parameters of temperature, pressure and flow rate of CO2 . Central composite design (CCD) based on response

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Figure 4. Schematic diagram of SFE process. Table 1. SC-CO2 extraction of phytosterols at different parameters from various plant matrices Extraction conditions Plant material

Pressure (MPa)

Flow rate of solvent

Temperature (∘ C)

Extraction time

Ref.

>0.26 >0.4 (99%)

𝛽-Sitosterol 𝛽-Sitosterol

12

3h

>0.6

Total phytosterols

22

9h 3h 6h

>0.26 >1.0 >1.25

𝛽-Sitosterol Total phytosterols 𝛽-Sitosterol, campesterol and stigmasterol – 𝛽-Sitosterol, campesterol, stigmasterol and 𝛽-amyrin >0.6 𝛽-Sitosterol, campesterol and stigmasterol >0.35 𝛽-Sitosterol, campesterol, stigmasterol and spinasterol 0.64 (average size) 𝛽-Sitosterol, stigmasterol, brassicasterol and campesterol – 𝛽-Sitosterol, stigmasterol, campesterol and Δ5 -avenasterol >0.85 𝛽-Sitosterol, stigmasterol and campesterol

78

15-60 15-45

40–80 35–65

Roselle seeds

20-40

40–80

Sea buckthorn seeds Kalahari melon seeds Grape seeds

15-60 20-40 37

40–80 40–80 65

0.8 g min−1 35–53 g g−1 of dried sample 10–20 mL min−1 (as a co-solvent, 2 mL min−1 of ethanol) 0.8 g min−1 10–20 mL min−1 60 g min−1

Lotus bee pollen

20–40

40–60

166.67–333.33 mL min−1

2.5 h

Rice bran

17–31

41.67 g min−1

6h

7.33 g min−1

4h

Pumpkin seeds

40

0–60 (two experiments in subcritical condition) 40

Tomato seeds

55.2

80

300 g min−1

2h

Amaranth seeds

31

50



16 h

Corn, sesame, oat and peanut

45

55

8000 mL min-1

2h

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Main analyzed compound

9h –

Sea buckthorn seeds Dandelion leaves

surface methodology (RSM) has been used to optimize the extraction conditions. The optimum conditions to maximize phytosterol concentration in the extract were a temperature of 40 ∘ C, a pressure of 30 MPa and a flow rate of CO2 of 12 mL min−1 . Among the extraction parameters, temperature greatly affects the phytosterol concentration in Kalahari melon seed oil. The concentration of

Particle size (mm)

21

79 80

81

82

83

84

91

92

total phytosterol decreased with an increase in temperature from 40 to 80 ∘ C. The increase in flow rate of CO2 also causes a slight reduction in phytosterol concentration in the extract. Nyam et al.22 also recovered phytosterol from roselle seeds using SC-CO2 with ethanol as co-solvent. In this study, authors optimized the three extraction parameters – pressure, temperature and flow

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Phytosterols and their extraction using supercritical carbon dioxide

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Figure 5. Extraction curves for 𝛽-sitosterol. Extraction conditions of runs 1–5: curve 1, 15 MPa, 40 ∘ C; curve 2, 30 MPa, 40 ∘ C; curve 3, 60 MPa, 40 ∘ C; curve 4, 30 MPa, 60 ∘ C; curve 5, 30 MPa, 80 ∘ C).12

rate of CO2 for the extraction of phytosterol-enriched oil using CCD based on RSM. The operating conditions for the extraction process were a pressure of 20–40 MPa, a temperature of 40–80 ∘ C and CO2 flow rate of 10–20 mL min−1 . Among the three parameters, pressure had the strongest effect on the concentration of phytosterol in roselle seed oil. From the experimental design, the authors reported that the optimum pressure, temperature and CO2 flow rate for phytosterol extraction were 40 MPa, 40 ∘ C and 20 mL min−1 , respectively. A further increase in solvent flow rate had no any significant effect on the yield of extract or phytosterol concentration. From a commercial point of view, the lowest possible level of CO2 should be used to recover a higher amount of oil enriched with phytosterol. The phytosterol concentration of a variety of grape seed oil obtained by SC-CO2 extraction has been reported by Beveridge et al.80 Authors performed the extraction only once, at a pressure of 37 MPa, a temperature of 65 ∘ C, CO2 flow rate of 60 g min−1 and extraction time of 6 h. The total phytosterol content of different grape seed oil samples varied from 4.10 to 18.62 mg g−1 oil. 𝛽-Sitosterol, campesterol, stigmasterol and Δ5 -avenasterol are common in all varieties. Among the phytosterols, 𝛽-sitosterol is the most important sterol in terms of quantity. Xu et al.81 performed SC-CO2 extraction of phytosterols from lotus bee pollen. The main sterol components of lotus bee pollen are 𝛽-sitosterol, campesterol and stigmasterol. The extraction conditions significantly affect the yield of total sterols. At low pressure, higher temperature causes a lower phytosterol yield due to the prominent effect of temperature on solvent density than on the vapor pressure of solute. Shen et al.82 analyzed the phytosterol concentration in rice bran oil extracted by SC-CO2 under various extraction conditions (pressure of 17–31 MPa, temperature of 0–60 ∘ C, CO2 flow rate of 2.5 kg h−1 and extraction time of 6 h). The authors also used CO2 to extract phytosterols under critical conditions. The effect of temperature and pressure on the extraction of the main phytosterols has been assessed with respect to the solvent consumed (Fig. 6). In most cases, the yield of phytosterols was higher with high pressure and moderate temperature. The yield of phytosterols was lower in subcritical conditions compared to critical conditions. Hrabovski et al.83 performed SC-CO2 extraction of phytosterol from pumpkin seed at a pressure of 40 MPa and a temperature of 40 ∘ C. It was reported that the total phytosterol content J Sci Food Agric (2014)

Figure 6. Extraction of phytosterol from rice bran by dense CO2 at different temperatures and pressures (CO2 flow rate, average of 2.5 kg h−1 ). (a) Stigmasterol; (b) campesterol; (c) 𝛽-sitosterol.82

of pumpkin seed oil was 2.94 mg g−1 oil. The most common plant sterols – campesterol, stigmasterol, 𝛽-sitosterol and Δ7 -avenasterol were present in pumpkin seed oil obtained by SC-CO2 extraction. In another study, phytosterols were extracted using SC-CO2 from tomato seed.84 The extraction was set to a temperature of 80 ∘ C, a pressure of 55.2 MPa and CO2 flow rate of 0.3 kg min−1 . In this study, the authors used high temperature and flow rate of CO2 to perform the phytosterol extraction. The

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www.soci.org concentration of total phytosterol in the extract was 12.30 mg g−1 oil, which was much higher than pumpkin seed. In most studies related to phytosterol extraction using SC-CO2 , the yield of phytosterol has been compared to the conventional organic solvent extraction process. Most authors reported that the amount of total phytosterol or individual phytosterols in the extract obtained by SC-CO2 extraction was higher than that of conventional organic solvent extraction.12,80,83 This proves that SC-CO2 extraction is efficient in extracting sterols from various plant matrices. The extraction of phytosterol using SC-CO2 is much faster than the oil (triglyceride), which means that phytosterol can be concentrated in the extract with a low volume of solvent consumption. On the other hand, high pressure and moderate temperature favor phytosterol extraction from various plant matrices using SC-CO2 . Therefore, phytosterols can be extracted through SFE under moderate extraction conditions and less use of solvent. A few research studies have also been performed to purify phytosterol esters from soybean oil deodorizer distillates by countercurrent extraction using SC-CO2 .85,86 In countercurrent extraction, CO2 flowed continuously from the bottom of a column and the liquid sample was pumped from the top of the column. Before extraction, the raw material was first subjected to enzymatic reactions to obtain a mixture consisting of fatty acid ethyl esters, tocopherols and phytosterol esters, with minor amounts of free fatty acids, squalene, free sterols and triacylglycerols. Phytosterol esters were then separated from this mixture by countercurrent extraction using SC-CO2 . The extraction was carried out at pressures of 20–28 MPa, temperatures of 45–55.7 ∘ C and solvent-to-feed ratios of 15–35 kg kg−1 . Free phytosterol extracted from oils is widely used in fortified foods and dietary supplements. Some products such as margarine, yoghurt, yoghurt drinks and orange juice containing plant sterols are commercially available on the market.87 However, as supplementing foods, the use of phytosterol obtained by SC-CO2 extraction can be attractive owing to solvent-free products as well as green processing technology.

EXPERIMENTAL DESIGNS USED IN OPTIMIZATION OF PHYTOSTEROL EXTRACTION CONDITIONS Many parameters in the SFE process are being considered to optimize for maximizing the yield of extract and the compound of interest. To evaluate the impact of a large number of parameters on the yield of extract, many experimental runs are required, which is not economically and practically feasible. Therefore, recently experimental designs have been used to optimize the extraction conditions in SFE for achieving high extraction efficiency with a minimum number of experimental trials.88,89 The most common experimental designs used in SFE are full factorial design, fractional factorial design, Plackett–Burman design as screening design, and Taguchi design, CCD and Box–Behnken design as optimization design.90 A few experimental designs have been applied to optimize the extraction conditions in connection with phytosterols. Xu et al.81 used a two-factor central composite rotatable design (CCRD) to optimize phytosterol extraction using SC-CO2 from lotus bee pollen. From the experimental design, the optimum condition was set to the pressure of 38.2 MPa and temperature of 49.7 ∘ C. Pressure and temperature were two independent factors and the yield of phytosterols was predicted one of four responses. The patterns of surface response clearly indicated that higher temperature at low pressure provide a lower phytosterol yield, which is

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critically described earlier. Nyam et al.22,79 used CCD based on RSM for the optimization of phytosterol extraction from Kalahari melon and roselle seeds. Three extraction parameters (pressure, temperature and solvent flow rate) have been considered for this experimental design. The experimental data and predicted data from the experimental design showed that pressure had a profound effect on phytosterol extraction from roselle seeds compared with temperature. Sovova et al.78 applied an equation-based model, a broken and intact cell model (BIC model) linked to phase equilibrium. There are three models: mass transfer from broken and intact cells, equilibrium extraction for separation factor and extraction from the particle surface with combined equilibrium and separation factor. Separation factor was correlated to the density of CO2 .

CONCLUSIONS Phytosterols have great health benefits especially in the reduction of heart disease risk. Therefore, much attention has been given to extracting phytosterols by a variety of processes. SFE is an efficient technology for extracting various bioactive compounds from different plant matrices. It provides tremendous advantages over conventional liquid solvent extraction, such as selectivity, rapidity, environmental friendliness, overall cost effectively and reliability for thermolabile compounds. In particular, SC-CO2 is very selective for phytosterol isolation. It is capable of extracting sterols from plant matrices very quickly with higher yields as compared to conventional solvent extraction. Despite the clear advantages of SFE using CO2 in phytosterol extraction, experimental data are still lacking regarding the effect of all operating parameters on the extraction process. However, optimization of all extraction parameters may further enhance the efficiency of SFE technology and it may provide support for commercialization of phytosterols in health and for other purposes.

ACKNOWLEDGEMENT This work was supported by Exploratory Research Grant Scheme ERGS13-028-0028.

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J Sci Food Agric (2014)

Phytosterols and their extraction from various plant matrices using supercritical carbon dioxide: a review.

Phytosterols provide important health benefits: in particular, the lowering of cholesterol. From environmental and commercial points of view, the most...
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