Extraction and Fractionation of

antioxidants and nutraceuticals using

innovative supercritical tecnologies
Idea abstract
Demand of food products with recognized benefits to health and wellness had increased the
interest of industry and scientific community to identify and to extract bioactive compounds from
natural matter and to develop the so called functional foods or nutraceuticals (1). A
nutraceutical is any substance that is a food or a part of a food and provides medical or health
benefits, including the prevention or treatment of disease. Such products may be isolated
nutrients, dietary supplements or genetically engineered foods, herbal products, or processed
foods such as cereals, soups and beverages (2).
In all food processing step by-products and wastes are generated in considerable quantities,
among them: seeds, skin, pulp, damaged fruits and water (4). Disposal of these by-products
and wastes represents an environmental and economic problem to the industry (5). In the
traditional extraction of bioactive compounds, there are some difficulties related to the use of
organic solvents: toxicity, low selectivity, pollution, low yield, high cost, large quantities of
solvent and raw material, and downstream processing to eliminate the solvent in the final
product (5).
In the last two decades innovative processes have been developed to improve the quality of
products and to reduce or eliminate the related pollution. Among these, supercritical fluid
extraction (SFE) is an innovative technology that allows to use gases at supercritical conditions
as a solvent. The extraction with supercritical fluids, particularly allows tuning solvent power and
selectivity of the gas used when pressure and temperature are modified over their critical
values. Even more, the gas employed as solvent for SFE does not represent a pollution factor
by the fact that is not generated in the process.
Different applications of SFE using carbon dioxide (CO2) have been developed to extract
lipophilic and non-polar compounds of low molecular weight. Nevertheless, the SFE using CO2
has limitations due to its affinity by non-polar compounds; it means that the solubility of polar
compounds in supercritical CO2 (SC-CO2) is low or near to zero. In order to easily fractionate
polar compounds, the supercritical antisolvente extraction (SAE) has been developed. In SAE
SC-CO2is used as antisolvent to recover or to fractionate bioactive compounds from an organic
solvent used in the extraction of bioactive compounds from natural matter.

Identified applications
The health benefits of nutraceuticals are in several areas, including cancer, diabetes, obesity,
atherosclerosis and other cardiovascular disease, aging process and immune responseenhancing effect, mental health. Benefic effects offered by nutraceuticals are due to a cocktail of
bioactive phytochemicals present in the products of interest. In Table 1 there are some
examples of functional compounds in many food sources.

Functionalcomponents

Source

Potentialbenefits

Carotenoids
Alpha-carotene/Betacarotene

Carrots, Fruits,
Vegetables

Neutralize free radicals, which may cause cell

damage

Lutein

Green vegetables

Reduce the risk of macular degeneration

Lycopene

Tomatoproducts
(ketchup, sauces)

Reduce the risk of prostate cancer

Insoluble Fibre

Wheat Bran

Reduce risk of breast or colon cancer

Beta-Glucan

Oats, barley

Reduce risk of cardiovascular disease. Protect

against heart disease and some cancers;
lower LDL and total cholesterol

Soluble Fibre

Psyllium

Reduce risk of cardiovascular disease. Protect

against heart disease and some cancers;
lower LDL and total cholesterol

Long chain omega-3 Fatty

Acids-DHA/EPA

Salmon and other fish

oils

Reduce risk of cardiovascular disease.

Improve mental, visual functions

ConjugatedLinoleicAcid
(CLA)

Cheese, meatproducts

Improve body composition. Decrease risk of

certain cancers

Anthocyanidins

Fruits

Neutralize free radicals; reduce risk of cancer

Catechins

Tea

Neutralize free radicals; reduce risk of cancer

Flavonones

Citrus

Neutralize free radicals; reduce risk of cancer

Flavones

Fruits/vegetables

Neutralize free radicals; reduce risk of cancer

Lignans

Flax, rye, vegetables

Prevention of cancer, renal failure

Tannins
(proanthocyanidines)

Cranberries, cranberry
products, cocoa,
chocolate

Improve urinary tract health. Reduce risk of

cardiovascular disease

DietaryFibre

FattyAcids

Phenolics

PlantSterols

Stanolester

Corn, soy, wheat, wood

oils

Lower blood cholesterol levels by inhibiting

cholesterol absorption

Fructo-oligosaccharides
(FOS)

Jerusalem artichokes,
shallots, onion powder

Improve quality of intestinal microflora;

gastrointestinal health

Lactobacillus

Yogurt, Otherdairy

Improve quality of intestinal microflora;

gastrointestinal health

Soybeans and soybased foods

Menopause symptoms, such as hot flashes. It

protects against heart disease and some
cancers; lower LDL and total cholesterol

Prebiotics/Probiotics

Soy Phytoestrogens
Isoflavones:
DaidzeinGenistein

Examples of functional or nutraceutical food components (6)

The mechanisms by which benefits of nutraceutical and functional foods in health are expressed
can vary; but, generally include antioxidant and anti-inflammatory activity or a combination of
them; the increase activity of enzymes that detoxify carcinogens, inhibition of N-nitrosamine
formation, change of estrogen metabolism and many others (7).
Looking for an efficient use of by-products, different studies propose the approach to fruit and
vegetable residues as new sources of bioactive compounds (3, 15-17); indeed, this kind of
wastes contain relevant quantities of bioactive compounds at lower cost, not only for their
application on food industry, but also in chemical, pharmaceutical, and cosmetic industry (18).
These studies concluded that by-products are a good source of nutraceutical compounds with
physiological (Table 2) activity like antioxidant, anti-inflammatory, anti-diabetes, anti-obesity and
others. In addition, the importance in using agroindustrial by-products is due to the high
availability and low or near zero cost of this raw material (16).
By-products of:

Guava: refining pulping

Different residues of fruits and
vegetables
Juices: beet, apple, strawberry and
pear
Canned: tomato, artichoke and
asparagus
Harvesting: chicory, endive,
cucumber, broccoli
Cocoa: powder, skin and shell.
Grape (red grape marc): seeds and
skins
Mango skins, roselle seed, soy,
cocoa skins, pulp of guava

Bioactive compounds

Reference

Phenols and flavonoids

(16)

Antioxidants
(17)
Antioxidants
(19)
Phenols, flavonoids, anthocyanins,
tannins, proanthocyanidins

(20)

Antioxidants

(21)

Nutraceutical compounds extracted from agroindustrial by-products

More specifically, individual compounds were studied in fruit by-products, some of them are
shown on Table 2.
By-products

Compounds

Apple skins

Catechins, hydroxycinnamates, quercetin glycosides, and procyanidins

Grape skins

Anthocyanins, catechins, epicatechinsflavonols glycosides, phenolic

acids

Mango peels

Gallic acid and ellagic acids, gallotannins and condensed tannins

Kiwi fruitsreject

Phenolic acids, flavonol monomers and oligomers, and flavonols

glycosides

Phenolic compounds extracted from fruit by-products, Schieber et al. (3)

Current approaches
Extraction techniques for nutraceuticals have been widely investigated to obtain valuable
compounds from plant stuff for commercialization and research purposes. Traditional methods,
such as soxhlet, hydrodistillation and maceration, have been used for many decades; however,
they are time consuming and require relatively large quantities of solvent and have a low
selectivity for several kind of compounds (22).
Soxhlet extraction is a standard, well-established technique, which overcomes in performance
other conventional extraction techniques except for the limitations for processing thermolabile
compounds used in food and pharmaceutical applications. The advantages of soxhlet extraction
are: the displacement of transfer equilibrium by repeatedly bringing fresh solvent into contact
with the solid matrix, the maintainance of a relatively high extraction temperature using heat
from the distillation flask, the reduced need of filtration. In contrast, the disadvantages are long
time consuming, large amount of solvent used, agitation cannot be provided, the solvent used
must be eliminated by evaporation/concentration with the possibility of thermal denaturation by
the high temperature of extraction related to the boiling point of solvent (23).
Therefore, there is a demand for new extraction techniques to reduce extraction time, organic
solvent consumption and prevent pollution. New methods have been proposed for extracting
chemical compounds in a fast and efficient way from solid plant matrix, some of these are (22):
sonication-assisted extraction
microwave-assisted extraction
supercritical fluid extraction
accelerated solvent extraction
When the technique has to be selected, for the extraction of nutraceuticals, some
considerations have to be done, such as, for example, characteristics of plant material, nature of
nutraceuticals, sample preparation, nature of solvents used. Sometimes the highest yield of
extraction does not ensure the higher quantity of bioactive components in the extract (5). Some
bioactive compounds, such as free fatty acids and tocopherols, are sensitive to oxygen and
heat, therefore the yield and quality of bioactive components should be considered.

Technology description
Supercritical fluids are systems formed by one or more compounds at conditions over their
critical values of pressure and temperature. At critical conditions, the fluid has some properties
typical of gas and some properties typical of liquid (24), these can be modulated changing
pressure and temperature and improving solvent characteristics.
Carbon dioxide (CO2) is the gas mostly employed at supercritical conditions, not only because
of the favorable critical conditions of pressure (7.36 MPa) and temperature (304.15 K), but also
because it is non toxic, non-flammable and has a low cost at high purity. Particularly, at the end
of extraction, the dense gas is depressurized and the product is recovered without or with a
minimum content of solvent residues (25).
Different applications of SFE using CO2 have been developed to extract lipophilic and non-polar
compounds at low molecular weight:
Decaffeination of green coffee (26)
Extraction of nicotine from tobacco (27)
Extraction of chemotherapeutic agents (28)

SFE from solid matrix

SFE consists of two steps: 1) extraction and 2) separation of solvent and extract (Figure 1). In
SFE, SC-CO2 flows through a fixed bed of the natural matrix and extracts all compounds
soluble at fixed operating conditions. The solution SC-CO2+solubilized components goes
through a reduction valve and arrives to a precipitator where the depressurization allows to
separate the solubilized components from SC-CO2; if the pressure is low enough, the CO2
flows as a gas and the extract is collected.
Basic scheme of SFE
In sophisticated processes, two or more separators are employed to perform fractionation of the
solubilized compounds; the fractionation can be performed at different operating conditions of
pressure and temperature (29), in order to collect different compounds.
The low dielectric constant of CO2 limits the solubility of compounds to non-polar (30), in this
case another modification of original process consists of adding lower quantities of a polar
solvent, that permits to improve the solvent power of CO2 for polar compounds (31). This
solution requires the addition of post processing to separate solubilized compounds from the
liquid solvent (liquid at room conditions) or to study the process operating conditions in order to
obtain a selected separation of the solvent by SC-CO2 itself. The solvent used should meet the
considerations of safety; for this reason, the most used solvent is ethanol (32).

SFE from liquid solutions

The supercritical antisolvent extraction (SAE) is a combination of SFE that uses the principle to
separate or fractionate a complex liquid mixture of compounds. Figure 2 shows a schematic
representation of SAE process. The system is based on two principles: 1) the solvent initially
used to obtain the liquid solution of compounds (extraction) must be completely solubilized in
SC-CO2 at operating conditions, and 2) the compounds extracted by the liquid solvent should
not be solubilized or partially solubilized in SC-CO2. The process consists in a chamber where
SC-CO2 continuously flows and liquid solution is fed inside, the high affinity of CO2 to liquid
solvent leads to the precipitation of solid compounds (not solubilized in CO2), while the solvent
is recovered in a separator at low pressure. A filter inside the chamber at defined porosity
retains solids. When the objective is to fractionate compounds, the flow at the exit of the

precipitator is introduced in a train of separators like in SFE, at different pressure conditions

(34), to collect compounds solubilized in the SC-CO2+ethanol solution.
Scheme of supercritical antisolvent extraction
The SAE technique has been used in few applications: in Table 3 some of them are listed.
Nevertheless, SAE technique has many potential applications in food, pharmaceutical and
chemical industry.
Initial mixture
Propolis tincture
Grape skin + methanol
Oat bran + acetone
Lecitin + triglyceride oil
Oat bran + ethanol
Diary by-products + water
Ryanodol + etanol

Compound to recovery

Reference

Flavonoids

(33)

Polyphenols and anthocyanins

(34)

Triacil glycerol

(35)

Lecitin

(36)

Triacylglycerols

(37)

Beta serum

(38)

Ryanodol

(39)

Processes using SAE technique

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