Research Activity on Supercritical Fluids Technology at Kumamoto
UniversitySupercritical CO2 Extraction/Fractionation and Hydrothermal Extraction ofValuable Compounds from Natural Products
As mention above, CO2 is the best supercritical fluid in the field of extraction ofheat-sensitive components mainly destined for human nutrition, due to its chemical andphysical properties and its low critical temperature and pressure values. Recently, water
has also been used as supercritical solvent for extraction, especially in the field of foodapplication. Water is polar component that environmentally friendly solvent, especiallyfor non heat-sensitive compounds.
Extracts obtained by means of supercritical fluid extraction are of higher quality
than those obtained by organic solvent extraction or by water and steam distillation,
which can induce thermal degradation, hydrolysis and water solubilization of somecomponents. In short, supercritical CO2 extraction offers high selectivity in theextraction compounds, shorter extraction period; no solvent residue, with positiveeffects on extract quality. Also, the flexibility in the control of the variables involved in
the supercritical fluids extraction process allows one to optimize the experimental
conditions, considering the manipulation of a substance of interest.
In our laboratory, valuable compounds including essential oil, carotenoids, fatty
acids, phenolic compounds and alkaloids have been extracted from various natural
products using supercritical CO2 and water at hydrothermal condition. Schematic
diagram of supercritical CO2 and hydrothermal extraction apparatus are shown in Figure
1 and 2, respectively. The apparatuses are mainly consisted of high pressure pumps,extractor with the heater, back pressure regulator, extract collector, chiller and gas meter
for CO2.

Back pressure valve

Extractor

T3T2P

Back pressurevalve

CO2

Solvent mixer

Chiller

Heat
Exchanger

Flow meterCollectionvial

CO2pump

T1Extractor

extract
Entrainerpump
CO2 cylinder

Entrainer
Solvent

Heatingchamber

Water
bath
Glass beads

Water
pump

Heating
chamber

Water

Figure 1. Schematic diagram ofsupercritical CO2 extraction apparatus.

Figure 2. Schematic diagram ofhydrothermal extraction apparatus.

Essential oils are mainly formed by hydrocarbon and oxygenated terpenes and byhydrocarbon and oxygenated sesquiterpenes. Usually, they are used as flavor in foodand beverage. From the point of view of supercritical fluids extraction, essential oil
isolation is an example of extraction plus fractional separation. Indeed, this process canbe optimally performed operating at mild pressures (from 9 to 20 MPa) andtemperatures (from 40 to 50oC) since at these process conditions all the essential oil

component are largely soluble in SC-CO2. Essential oil from sunflower seeds [2],geranium [3], nutmeg seeds [4], Turkish plants [5] and Japanese pepper [6] have been
extracted with supercritical CO2. Composition and yield of extracted essential oils wereanalyzed qualitatively and quantitatively using GC-MS and GC-FID, respectively.
Optimum extraction condition of each material was also reported. We also developed
fractionation of essential oils (bergamot [7-8], citrus oil [9], lemon oil [10] and yuzu oil[11]) using supercritical CO2 to selectively separate a valuable compound. Moreover,fractionation of α-tocopherol from oil using supercritical CO2 was also investigated[12].
Vegetable oils are a natural, convenient source of such unsaturated fats. Vegetableoil is usually produced from ground seeds. We have presented supercritical CO2
extraction of oil from seeds [13-15]. Seed oils mainly formed by triglycerides arereadily soluble in supercritical CO2 at 40oC and at pressure larger than 20 MPa. The
main parameters for this process are particle size, pressure and extraction time. Small
particles and high pressures can increase extraction rate in order to reduce extraction
time. In some cases, after extraction process, the supercritical CO2 triglycerides solution
is separated in the separator at sub-critical conditions to reduce to near zero the CO2
solvent power and allow the recovery of oil. Furthermore, oil free gaseous CO2 isobtained in the separator.
Carotenoids are the most famous one of the nutraceuticals. Carotenoids are afamily of compounds that possess antioxidant and colouring properties for food,cosmetic and medical applications. They can be obtained in a large variety of natural
sources, such as vegetables, animals, yeasts and microalgae. Carotenoids have very low
or moderate solubilities in supercritical CO2. Therefore, in several cases supercritical
CO2 has been added with a co-solvent. The most frequently used co-solvent is ethanol
since its presence in the extracts does not compromise the use in food applications.
However, it is better, when it is possible, to avoid the use of a co-solvent that tends toregenerate the problem of solvent elimination from the extracts. In our group,
carotenoids, such as β-carotene, lycopene, astaxanthin and lutein, have been extracted
from tomato pulp [16], microalgae [17-20] and rosehip [21], using supercritical CO2
with or without co-solvent. Carotenoids have been successfully extracted with highyield and selectivity. Optimization of process, extraction rate and solubility ofcarotenoids in supercritical CO2 were reported. Generally, carotenoids yield strongly
increased with increasing temperature due to their localization in the plants matrix.
Polyphenolic compounds are containing a polyphenolic substructure that hasmedicinal properties. The main source of polyphenol is nutritional, since they are foundin a wide array of phytonutrient-bearing foods, such as fruits and vegetables.
Polyphenolic compounds are separated into two categories flavonoids andnon-flavonoids. Flavonoids include anthocyanins and tannins which contribute to thecolor and mouthfeel of fruits and vegetables, which have violet color. Non-flavonoids
include stilbenes such as resveratrol and compounds derived from acids in fruits andvegetables like benzoic, caffeic and cinnamic acid. In grapes, phenolics are foundwidely in the skin, stems and seeds. They are water soluble and will often secrete into
the vacuole of grape berries as glycosides. However, they have very low solubilities insupercritical CO2. Furthermore, addition of co-solvent with high polarity is necessary toenhance CO2 power. We have investigated polyphenolic compounds having antioxidant
activity extraction from various plants using supercritical CO2 with co-solvent andwater at hydrothermal condition [22-25]. Moreover, other extraction methods were also

developed to obtain higher extraction yield of polyphenolic compounds [26-27]. Except
polyphenolic compounds, extraction of other biological active compounds from variousplants, such as Turmeric [28], biwa seeds [29], rice bran [30], citrus peel [31] andmushroom [32], were also investigated.
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