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3 Isolation of essential oil from different plants and herbs
4 by supercritical fluid extraction
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8 Tiziana Fornari*, Gonzalo Vicente, Erika Vázquez, Mónica R. García-
Risco, Guillermo Reglero
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13 Instituto de Investigación en Ciencias de la Alimentación CIAL (CSIC-UAM).
14 CEI UAM+CSIC. C/Nicolás Cabrera 9, Universidad Autónoma de Madrid,
15 28049 Madrid, España.
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20 * Corresponding author: Instituto de Investigación en Ciencias de la Alimentación CIAL
21 (CSIC-UAM). C/ Nicolás Cabrera 9. Universidad Autónoma de Madrid. 28049, Madrid,
22 Spain Tel: +34661514186. E-mail address: tiziana.fornari@uam.es
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*Manuscript
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24 Abstract
25 Supercritical fluid extraction (SFE) is an innovative, clean and environmental friendly
26 technology with particular interest for the extraction of essential oil from plants and herbs.
27 Supercritical CO2 is selective, there is no associated waste treatment of a toxic solvent, and
28 extraction times are moderate. Further supercritical extracts were often recognized of superior
29 quality when compared with those produced by hydro-distillation or liquid-solid extraction.
30 This review provides a comprehensive and updated discussion of the developments and
31 applications of SFE in the isolation of essential oils from plant matrices. SFE is normally
32 performed with pure CO2 or using a cosolvent; fractionation of the extract is commonly
33 accomplished in order to isolate the volatile oil compounds from other co-extracted
34 substances. In this review the effect of pressure, temperature and cosolvent on the extraction
35 and fractionation procedure is discussed. Additionally, a comparison of the extraction yield
36 and composition of the essential oil of several plants and herbs from Lamiaceae family,
37 namely oregano, sage, thyme, rosemary, basil, marjoram and marigold, which were produced
38 in our supercritical pilot-plant device, is presented and discussed.
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46 Keywords: supercritical extraction; carbon dioxide; essential oil; Lamiaceae plants;
47 bioactive ingredients.
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49 Contents
50 1. Introduction
51 2. The essential oil of plants and herbs
52 3. Supercritical fluid extraction (SFE) of essential oils
53 3.1 Effect of matrix pre-treatment and packing
54 3.2 Effect of extraction conditions
55 3.3 Fractionation alternatives
56 3.4 Ultrasound assisted SFE
57 4. Supercritical chromatography fractionation of essential oils
58 5. Comparison of the SFE extraction of essential oil from different plant matrix
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60 1. Introduction
61 Essential oils extracted from a wide variety of plants and herbs have been traditionally
62 employed in the manufacture of foodstuffs, cosmetics, cleaning products, fragrances,
63 herbicides and insecticides. Further, several of these plants have been used in traditional
64 medicine since ancient times as digestives, diuretics, expectorants, sedatives, etc., and are
65 actually available in the market as infusions, tablets and/or extracts.
66 Essential oils are also popular nowadays due to aromatherapy, a branch of alternative
67 medicine that claims that essential oils and other aromatic compounds have curative effects.
68 Moreover, in the last decades, scientific studies have related many biological properties
69 (antioxidant, anti-inflammatory, antiviral, antibacterial, stimulators of central nervous system,
70 etc.) of several plants and herbs, to some of the compounds present in the essential oil of the
71 vegetal cells [1-5]. For example, valerenic acid, a sesquiterpenoid compound, and its
72 derivatives (acetoxyvalerenic acid, hydroxyvalerenic acid, valeranone, valerenal) of valerian
73 extract are recognized as relaxant and sedative; lavender extract is used as antiseptic and anti74
inflammatory for skin care; menthol is derived from mint and is used in inhalers, pills or
75 ointments to treat nasal congestion; thymol, the major component of thyme essential oil is
76 known for its antimicrobial activity; limonene and eucalyptol appear to be specifically
77 involved in protecting the lung tissue. Therefore, essential oils have become a target for the
78 recovery of natural bioactive substances. For example, nearly 4000 articles in which
79 “essential oil” or “volatile oil” appears as keyword were published in the literature since year
80 2000 up today (http://www.scirus.com/); around 3000 also include the word “bioactive” or
81 “bioactivity” in the article text.
82 Essential oils are composed by lipophilic substances, containing the volatile aroma
83 components of the vegetal matter, which are also involved in the defense mechanisms of the
84 plants. The essential oil represent a small fraction of plant composition, and is comprised
85 mainly by monoterpenes and sesquiterpenes, and their oxygenated derivatives such as
86 alcohols, aldehydes, ketones, acids, phenols, ethers, esters, etc. The amount of a particular
87 substance in the essential oil composition varies from really high proportions (e.g. around 80-
88 90 %w/w of δ-limonene is present in orange essential oil) to traces. Nevertheless,
89 components present in traces are also important, since all of them are responsible for the
90 characteristic natural odor and flavor. Thus, it is important that the extraction procedure
91 applied to recover essential oils from plant matrix can maintain the natural proportion of its
92 original components [6].
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New effective technological approaches to extract 93 and isolate these substances from raw
94 materials are gaining much attention in the research and development field. Traditional
95 approaches to recover essential oil from plant matrix include steam- and hydro-distillation,
96 and liquid-solvent extraction. One of the disadvantages of steam-distillation and hydro97
distillation methods is related with the thermolability of the essential oil constituents, which
98 undergo chemical alteration due to the effect of the high temperatures applied (around the
99 normal boiling temperature of water). Therefore, the quality of the essential oil extracted is
100 extremely damaged [6].
101 On the other side, the lipophilic character of essential oils requires solvents such as paraffinic
102 fractions (pentane and hexane) to attain an adequate selectivity of the extraction. Further,
103 liquid solvents should have low boiling points, in order to be easily separated from the extract
104 and re-utilized. In this sense, the main drawback is the occurrence of organic toxic residues in
105 the extracted product.
106 Among innovative process technologies, supercritical fluid extraction (SFE) is indeed the
107 most widely studied application. In practice, SFE is performed generally using carbon
108 dioxide (CO2) for several practical reasons: CO2 has moderately low critical pressure (74 bar)
109 and temperature (32C), is non-toxic, non-flammable, available in high purity at relatively
110 low cost, and is easily removed from the extract. Supercritical CO2 has a polarity similar to
111 liquid pentane and thus, is suitable for extraction of lipophilic compounds. Thus, taking into
112 account the lipophilic characteristic of plant essential oils, it is obvious that SFE using CO2
113 emerged as a suitable environmentally benign alternative to the manufacture of essential oil
114 products.
115 The commercial production of supercritical plant extracts has received increasing interest in
116 recent decades and has brought a wide variety of products that are actually in the market. As
117 mentioned before, supercritical plant extracts are being intensively investigated as potential
118 sources of natural functional ingredients due to their favorable effects on diverse human
119 diseases, with the consequent application in the production of novel functional foods,
120 nutraceuticals and pharmacy products. The reader is referred to several recent works [7-10] in
121 which is reviewed the supercritical extraction and fractionation of different type of natural
122 matter to produce bioactive substances. The general agreement is that supercritical extracts
123 proved to be of superior quality, i.e. better functional activity, in comparison with extracts
124 produced by hydro-distillation or using liquid solvents [11-14]. For example, Vági et al. [11]
125 compared the extracts produced from the extraction of marjoram (Origanum maorana L.)
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using supercritical CO2 (50ºC and 45 MPa) and ethanol Soxhlet 126 extraction. Extraction yields
127 were, respectively, 3.8 and 9.1%. Nevertheless, the supercritical extract comprised 21% of
128 essential oil, while the alcoholic extract contained only 9% of the volatile oil substances.
129 Furthermore, studies related with the antibacterial and antifungal properties of the extract
130 revealed better activity for the supercritical product. Another example of improved biological
131 activity exhibit by supercritical extracts was reported by Glisic et al. [14], demonstrating that
132 supercritical carrot essential oil was much more effective against Bacillus cereus than that
133 obtained by hydro-distillation.
134 Indeed, numerous variables have singular effect on the supercritical extraction and
135 fractionation process. Extraction conditions, such as pressure and temperature, type and
136 amount of cosolvent, extraction time, plant location and harvesting time, part of the plant
137 employed, pre-treatment, greatly affect not only yield but also the composition of the
138 extracted material.
139 Knowledge of the solubility of essential oil compounds in supercritical CO2 is of course
140 necessary, in order to establish favorable extraction conditions. In this respect, several studies
141 have been reported [15-18]. Nevertheless, when the initial solute concentration in the plant is
142 low, as is the case of essential oils, mass transfer resistance can avoid that equilibrium
143 conditions are attained. Therefore, pretreatment