gamma-linolenic and stearidonic acids: purification and upgrading of c18-pufa oils

Review Article

Gamma-linolenic and stearidonic acids:Purification and upgrading of C18-PUFA oils

Jose Luis Guil-Guerrero, Miguel Angel Rincon-Cervera and Elena Venegas-Venegas

Food Technology Division, University of Almeria, Almerıa, Spain

Keywords: C18-PUFA / Essential fatty acid / Fatty acid purification / g-Linolenic acid / Stearidonic acid

Received: December 26, 2009 / Revised: May 1, 2010 / Accepted: June 10, 2010

DOI: 10.1002/ejlt.200900294

1 Introduction

Polyunsaturated fatty acids (PUFAs) are a group of fatty

acids (FAs) containing two or more double bonds between

carbon atoms in their chain. There are two families of PUFAs

(depending on the position of the double bonds in the chain)

which are essential for human health. One of them is the n-3

family, also called v-3 family, which recently has received

much attention because of its various physiological functions

in the human metabolism. Fish oil contains noticeable

amounts of eicosapentaenoic acid (EPA, 20:5n-3), docosa-

hexaenoic acid (DHA, 22:6n-3), and minor quantities of

stearidonic acid (SDA, 18:4n-3), while plant seed oils, such

as flax oils, contain a-linolenic acid (ALA, 18:3n-3). The

other family is named n-6 (v-6) and includes linoleic

acid (LA, 18:2n-6), arachidonic acid (AA, 20:4n-6), and

g-linolenic acid (GLA, 18:3n-6) [1]. LA and ALA are con-

sidered essential FAs (EFAs) and their absence in a normal

diet has been described as responsible for the development of

a wide range of diseases such as cardiovascular disorders,

inflammatory processes, viral infections, certain types of can-

cer, and autoimmune disorders [2, 3]. LA and ALA can be

metabolized to AA and DHA, respectively, by the consecu-

tive action of desaturases and elongases. Both n-3 and n-6

PUFAs are precursors of hormone-like compounds, the

eicosanoids (prostaglandins, thromboxanes and leuko-

trienes), which are involved in many important biological

processes in the human body [4, 5].

The therapeutic and preventive benefits of dietary n-3 FAs

with regard to cardiovascular disease and rheumatoid arthritis

have been well documented, and most evidence for benefits

applies to the long-chain n-3 FAsEPAandDHA,which can be

found mainly in fish tissues [6, 7]. However, for many people

whowish to obtain the healthy benefits provided by dietary n-3

FAs, daily ingestion of fish or fish oil is not a sustainable long-

term approach because of pressure on global fish stocks, and

aquaculture is also unlikely to be a proper solution because the

industry relies heavily on wild fish stocks for feed. These issues

could be resolved by the provision of n-3 FAs via the terrestrial

food chain. To increase the number of suitable dietary options,

a land-based source of n-3 FAs capable to be effective in

increasing tissue concentrations of the long-chain n-3 EPA

and DHA is required [8]. Currently, ALA is the main n-3 FA

available in vegetal oils. However, there is poor conversion of

ingested ALA to longer-chain n-3 FAs as EPA and DHA [9]

because the initial enzyme in the metabolic pathway, D6-desa-

turase, which converts ALA to SDA, is rate limiting in humans

[10]. This enzyme is the same that acts to convert LA to GLA

[11]. Thus, the ingestion of vegetal oils enriched in SDA and

GLA (Fig. 1) could be an efficient way to minimize the limited

action of D6-desaturase, which is hindered by several factors,

including aging, nutrient deficiency, smoking, and excessive

alcohol consumption [12]. It has been shown that conversion

of dietary SDA into EPA in human erythrocytes and plasma

phospholipids is more effective than conversion of dietary

ALA: whereas 1 g dietary SDA is approximately equivalent

to 300 mg dietary EPA, it is necessary to eat 4.3 g ALA to

reach the same amount of tissue concentration of EPA [8]. So,

it would be interesting to attempt the purification of triacyl-

glycerol (TAG) species containing GLA and SDA by using

natural sources as raw material in order to use them with

alimentary or pharmaceutical purposes.

Correspondence:Dr. Jose Luis Guil-Guerrero, Food Technology Division,

University of Almeria, 04120 Almerıa, Spain

E-mail: [email protected]

Fax: 34-950015484

Abbreviations: AA, arachidonic acid; ALA, a-linolenic acid; CPC,

centrifugal partition chromatography; DHA, docosahexaenoic acid;

EPA, eicosapentaenoic acid; FA, fatty acid; FFA, free FA; GLA, g-

linolenic acid; LA, linoleic acid; PUFA, polyunsaturated fatty acid;

SDA, stearidonic acid; SFE, supercritical fluid extraction; TAG,

triacylglycerol

1068 Eur. J. Lipid Sci. Technol. 2010, 112, 1068–1081

� 2010 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim www.ejlst.com

Under certain conditions, such as decreased enzymatic

activity of D6-desaturase, GLA may become conditionally

essential [12]. This FA exhibits anti-inflammatory, antith-

rombotic, and lipid-lowering potential. It also enhances

smooth muscle relaxation and vasodilatation. In addition,

all EFAs including both GLA and SDA are important con-

stituents of membrane phospholipids, where they enhance

the integrity and the fluidity of the same one [12].

This paper is focused to relate and compare the different

procedures to obtain GLA and SDA, which seems to be the

more demanded PUFAs for the future, by considering the

above exposed reasoning. Usually, most of the purification

procedures for both FAs are similar, and some of the reviewed

techniques yield both ones simultaneously.

2 Extraction, upgrading, and purification ofC18 PUFA-containing oils

2.1 Gamma-linolenic and stearidonic acid sources

GLA is found naturally in the TAG fractions of some plant

seed oils. The richest sources of GLA include evening prim-

rose oil, borage oil, blackcurrant oil, and hemp seed oil [1].

GLA is also found in some fungal sources, and a minimal

amount is produced in the human body as a downstream

metabolite of the D6-desaturase induced conversion from LA

[1]. GLA is present in its natural sources in variable amounts,

being one of its richest source borage (Borago officinalis L.)

seed oil (20–25% GLA on total FAs) [1]. Evening primrose

(Oenothera biennis L.) seed oil has been frequently used to

purify GLA: although its GLA percentage is not the highest

(8–14%), it is easily available and shows a simple FA and

TAG composition, which makes it easier to purify GLA from

this source.

SDA is present in some species of algae, fungi, and seed

oils, and also in some species of boraginaceae and primula-

ceae [11]. Studies about distribution and isolation of SDA

from natural sources began with works of Seylers [13], who

reported the presence and isolation of SDA in herring oil.

Simultaneously, Klenk and Brockerhoff [14] reported the

isolation and structure of an octadecatetraenoic acid from

South African pilchard oil. Later on, SDA is reported and

isolated from the seeds of Lithospermum officinale [15].

Recently, the SDA content in TAG species from Echium

vulgare L. has been reported [16]. Another option to get a

SDA-enriched oil could be achieved by conducting genetic

modifications in appropriate oilseed plants; this way, a genet-

ically modified soybean oil containing �20% SDA has been

reported as a potential source of SDA [17].

Methods for GLA and SDA concentration are similar to

those employed for other FAs, but just few of them are

suitable for large-scale production. Each technique has its

own advantages and drawbacks. The following provides a

background to each of these methods. Few of the methods

described in this paper are now used for industrial scale

production with variable contents of PUFAs. The challenge

now is how to develop cost-effective methodologies to pro-

duce PUFA concentrates to meet the growing demand.

2.2 C18-PUFA oil extraction

2.2.1 Extraction by classic procedures

The process to obtain FAs from seeds comprises several steps

in which diverse separation techniques and hydrolysis-sap-

onification reactions are involved. The fact of working with

lipids demands that the process must be quick and reliable, to

minimize the degradations and peroxidations [18]. Taking

into account that both GLA and SDA could be used in

functional foods and in the pharmaceutical or dietary indus-

try, the solvents used should be selected keeping inmind their

possible toxicity, handling easiness, security, and cost. So,

only biocompatible and legally accepted solvents should be

used when processing oils for these purposes [19]. In the

selection of the solvent type, it is also necessary to consider

the lipid type (polar or apolar) contained in the sample, as

well as the distribution among the different fractions of the

FAs to extract. For this reason, it is interesting to carry out a

previous lipid fractionation in neutral lipids (TAGs) and

polar lipids (glycolipids and phospholipids). A recent

example of SDA enrichment by means of lipids fractionation

is given for the marine macrophyte Laminaria japonica [20].

In algae harvested in winter, SDA reaches 54.3% in the

monogalactosyldiacylglycerol fraction, a considerable per-

centage suitable to continue the SDA purification processes.

Prior to lipids extraction, it is necessary to keep in mind

lipidic and nonlipidic components acting in cell interaction,

thus safe reagents for breaking these connections and avoid-

ing lipids degradation are necessary [21].

Hexane is usually chosen to extract seed oils, and also

petroleum ether or chloroform, having the last one a high

extraction yield, although it is a toxic solvent that has been

prohibited in the alimentary industry. Hexane at high con-

centrations has a well-known neurotoxic effect. However, it

seems to be not very toxic when it is used to laboratory scale

B) A)

GLA (18:3n-6) SDA (18:4n-3)

O

OH

O

OH

Figure 1. Molecular structures of GLA (A) and SDA (B).

Eur. J. Lipid Sci. Technol. 2010, 112, 1068–1081 Gamma-linolenic and stearidonic acids 1069


and it does not constitute a risk after evaporation [22]. The

first step, lipid separation of the seeds, is carried out bymeans

of solid–liquid extraction that basically consists in the extrac-

tion of a solute (lipidic fraction) included in the seeds by

means of an organic solvent. It is important to carry out the

whole extraction operation in a similar way that the final

purification of FAs: using inert atmosphere of nitrogen or

argon to prevent the lipid peroxidation [21]. For the same

reason, the operation should also be accomplished avoiding

light exposure, especially to the artificial light.

Some authors vary solvent and/or extraction method-

ology: MacKenzie et al. [23] used boiled isopropanol for seed

lipid extraction fromOnosmodium hispidissimum. Vioque et al.

[24] used hexane for seed lipid extraction from plant seeds.

Sridhar and Lakshinarayana [25] employed Kates method

[26], with chloroform-methanol 1:2 v/v for leaf lipid extrac-

tion. Daun and Tkachuk [27] extracted total lipids from wild

plant seeds with diethyl ether.

Although the lipidic extracts containing purified FAs can

be directly employed in some applications, to continue the

FAs purification process, the saponification of the extract is a

necessary step to obtain the free FAs (FFAs). The extractant

system usually used is composed by an alcohol and sodium

hydroxide (NaOH). After this, hydrochloric acid is added to

Figure 2. SDA (A) and GLA (B) extraction from two seeds by using several extracting systems. SFE-CO2 operating conditions were:

pressure, 300 bar; temperature, 408C; solvent flow rate, 1.5 L/min (STP); and extraction time, 3 h. GLA and SDA purity are shown as % on

total FAs.

1070 J. L. Guil-Guerrero et al. Eur. J. Lipid Sci. Technol. 2010, 112, 1068–1081


separate the FFAs that are recovered by means of a second

extraction, this time a liquid–liquid extraction (with an

organic solvent as n-hexane or diethyl ether).

Once FFAs are obtained, they should be preserved from

the peroxidation during storage. If samples will be stored

during relatively short periods of time, an inert solvent

must be used as preservant. Hexane has been shown to

be the best option among several solvents tested [18]. In

addition, the peroxidation rate depends both of the

temperature and the light; therefore, the samples must

be conserved at the lowest possible temperature and in

the darkness [28].

The direct saponification of the seeds to extract FFAs is

an alternative to obtain the lipid extract that can be carried

out with a 0.15%NaOH solution in 95% ethanol [29]. Other

authors have used KOH added to different extraction

mixtures for direct saponification. For example, to obtain

SDA from Isochrysis galbana biomass a hexane/ethanol

96% (1:2.5 v/v) KOH-containing mixture was used for direct

saponification [30].

Recently, SDA-oils extraction has been accomplished by

using several extracting solvent systems by our Research

Group [31]. CO2-supercritical fluid extraction (CO2-

SFE) as well as pure and classic mixtures of organic solvents

have been assayed to extract C18-PUFAs. As it is shown in

Fig. 2A, higher SDA purities were achieved by ethanol-

based solvent systems, probably due to the high polarity

of the SDA molecule. In contrast, higher GLA purities

(Fig. 2B) were obtained by using more apolar solvent

systems, SFE or simultaneous saponification-extraction

with ethanol as solvent.

Thus, taking into account the biocompatibility of

ethanol and other ecological considerations, ethanol-

based solvents seem to be the best option to extract

GLA and SDA oils from seeds. In addition, oil yield

after extraction processes were similar among all tested

extracting systems.

2.2.2 Extraction by solubility differences

The method is based on the different solubility properties of

sodium salts of FAs in ethanol, thus allowing FA concen-

tration from oils. Therefore, FAs having the same unsatura-

tion degree at room temperature, the shorter the hydrocarbon

chain and the higher the number of double bonds in the FA

molecule, the higher the solubility of the FA sodium salts is in

ethanol.

The possibility to extract directly a PUFA concentrate

from seeds was tested in Echium fastuosum, B. officinalis, and

Anchusa azurea seeds, which are a rich source of GLA, but

E. fastuosum has also noticeable amounts of SDA [32]. The

process was carried out through one single and ecological

step: simultaneous seed oil extraction/saponification/GLA

concentration [33]. The solubility difference method has

been employed as a previous step for enzymatic purification

processes. Although this procedure yields satisfactory results

for GLA, SDA was poorly concentrated (Fig. 3). This is due

to the fact that the variables of the process, such as tempera-

ture, were optimized for GLA concentration instead of SDA.

Thus, once optimized, this method should be satisfactory

employed for SDA concentration.

2.2.3 Supercritical fluid extraction

SFE is a relatively new separation process that lacks of some

of the drawbacks associated with the employment of conven-

tional separation techniques. A number of gases are known to

possess desirable selective solvent properties when raised to

temperatures and pressures above their critical values [34].

Usually, for oily seeds, CO2 is chosen because it has a mod-

erate critical temperature and pressure (304 K, 7.38 MPa)

being also inert, inexpensive, non-flammable, environmentally

acceptable, readily available, and safe [35]. PUFA separation

by SFE is related to the molecular size of the components

involved rather than their degree of unsaturation; therefore, a

Figure 3. E. fastuosum-FFAs profile obtained

by applying the solubility difference method at

48C. a FAs profile of E. fastuosum seed oil

obtained through the direct methylation of the

biomass. b Echium-FFA purities (%FA area on

total FA area detected by GLC) obtained after

applying the solubility difference method. c

Echium-FFA yields (%) reached after applying

the solubility difference method. FAs, fatty

acids; FFAs, free fatty acids; GLC, gas LC;

SAT, saturated; MON, monounsaturated; LA,

linoleic acid; GLA, g-linolenic acid; ALA,a-lino-

lenic acid; SDA, stearidonic acid.



prior concentration step is needed to achieve a high concen-

tration of PUFA in the final product [35]. Oils to be used for

PUFA concentration by SFE require preparation steps of

extraction, hydrolysis, and esterification by conventional

methods [36, 37].

Some of the processes used as pre-concentration step

previous to the supercritical CO2 extraction process are urea

fractionation, enzymatic fractionation, or industrial fermen-

tation of micro-organisms [38]. The hydrolysis of blackcur-

rant oil TAGs to produce FFAs with low content in GLA and

partial glycerols enriched in GLA has been reported using

supercritical CO2 [27].

The use of supercritical fluids for oil extraction and con-

centration of PUFA including GLA from fish oil, seaweed

[34, 35, 39, 40], and seed oils has been reported (Table 1)

[41–44] although pure or enriched fractions of SDA obtained

by this method have not been still documented. However,

further research will be required to determine the extent of its

use for SDA separation from oily sources. On the other hand,

the use of extremely high pressures and high capital costs

might limit the widespread use of this method to industrial

scale production.

2.3 C18-PUFA upgrading and purification

2.3.1 Urea complexation

This old method is based on the fact that the urea crystallizes

in a tightly packed tetragonal structure with channels of

5.67 A diameter. However, in the presence of long

straight-chain molecules it crystallizes in a hexagonal struc-

ture with channels of 8 � 1.2 A diameter within the hexag-

onal crystals [45]. Long-chain unbranched molecules are

retained in the channels, which are sufficiently large to

accommodate them.

The straight-chain of saturated FAswith six ormore carbon

atoms are readily adducted, but the presence of double bonds

in the carbon chain increases the bulk of the molecule and

reduces the likelihood of its complexationwith urea [46].Thus,

monoenes are more readily complexed compared to dienes,

and these are more readily complexed than trienes.

Therefore, formation of urea inclusion compounds

depends on the degree of unsaturation of the FAs. To

develop this process, the oil must be saponified and acidi-

fied to obtain FFAs using alcoholic KOH or NaOH, and

later on HCl. Simultaneously, unsaponifiables such as

sterols, vitamins A and D, and xenobiotics (e.g., PCB) as

well as other undesirable components are removed from it.

Finally, FFAs are mixed with an alcoholic (methanol or

ethanol) solution of urea and then allowed to cool to a

particular temperature depending on the degree of concen-

tration desired. Usually, a urea/FA ratio of 4:1 w/w is

used [47] (Table 2).

The urea fractionation method has been widely applied

and reported as a suitable procedure to separate EPA from

other FFAs in fish and microalgae oils [47]. This method has

shown to be effective to purifyGLA fromRibes nigrum seed oil

[48], being noticeable that the method was useful to separate

GLA from its positional isomer ALA, which is also present in

R. nigrum seed oil. Later on, the method was applied to

A. azurea, E. fastuosum, and S. sciophila seed oils [49]. It

was noted in these experiments that GLA percentages in

purified fractions increased when SDA does not appear as

a component in the oil. The most representative results when

concentrating GLA and/or SDA by this procedure are shown

in Table 2.

The urea fractionation method has also been successfully

used to get a GLA concentrate from borage oil, by means of

factorial experimentation response surface methodology for

optimizing the process conditions, accomplishing a concen-

trated fraction with 91.5% GLA [50]. On the other hand,

GLA from Spirulina platensis biomass, by using the galacto-

lipid fraction of the oil (39.0% GLA) has been concentrated

up to 90% purity [51].

The obtaining of a purified SDA fraction and other

PUFAs by urea complexation method was attempted

employing PUFAs from the marine microalga I. galbana

[47], a microalgae that contain 6.3% SDA in biomass, reach-

ing poorly result, because SDA as FFA percentage in the urea

concentrate was only increased until 22.6%.

Thus, the experience shows that the main difficulty to

reach a high SDA purity is the simultaneous presence in the

Table 1. Oil extraction yield and GLA amount from several oils using SFE

Oil

Extraction

parameters

Oil extraction yield

(wt% on dry biomass)

GLA

(% on total FAs)

Fungal oil from Cunninghamella echinulata [41] CO2, 300 bar, 508C 26.4 9.9

Borago officinalis seed oil [42] CO2, 300 bar, 408C 31.0 16.9




Oenothera biennis seed oil [42] CO2, 300 bar, 408C 21.0 3.7

Oenothera biennis seed oil [42] CO2, 200 bar, 608C 15.0 6.8



seed oils of other PUFAs such as GLA and ALA that co-

concentrate with the desired SDA. Unfortunately, the find of

SDA sources lacking of GLA and ALA seems to be unlikely,

because SDA is produced in a metabolic pathway in which

other C18-PUFAs are previously biosynthesized [11].

Nevertheless, this procedure could offer better results by

using SDA sources with a lower contain of others C18-

PUFAs, so single cell oils seem to be a promising alternative.

Nevertheless, for FA fractionation with alimentary pur-

poses, urea complexation method should be avoided, con-

sidering that the reaction of urea with ethanol or methanol

produces ethyl or methyl carbamates which have carcino-

genic properties [52].

2.3.2 Low-temperature crystallization

Winterization is a process that involves the chilling of the oil

to allow the solid portion to crystallize and the subsequent

filtration of the two phases [53]. The term ‘‘winterization’’

was originally applied decades ago when seed oil was sub-

jected to winter temperatures to accomplish the process of the

removal of solids by controlled crystallization and filtration. It

is a form of fractionation or the removal of solids at selected

temperatures. It involves the removal of a small quantity of

crystallized material from edible oils by filtration to avoid

clouding of the liquid fractions at refrigeration temperatures.

In a common procedure, the oil is chilled slowly to about 68Cduring a 24-h period. Cooling is stopped and the oil/crystal

mixture is allowed to stand for 6–8 h. The oil is filtered,

yielding 75–80% liquid oil [54]. Solvent winterization

involves the crystallization of desired fractions from oil dis-

solved in a suitable solvent. Fractions may be selectively

crystallized at different temperatures, separated and the sol-

vent removed for a final TAGmixture with a specific melting

point, TAG solubility or FA composition [54, 55].

Recently, solvent winterization of seed oil and FFAs was

employed to obtain GLA and SDA concentrates from the

seed oil of a Boraginaceae species, E. fastuosum. Different

solutions of seed oils and FFAs at 10, 20, and 40% w/w were

crystallized at 4,�24, and�708C, respectively, using hexane,

acetone, diethyl ether, isobutanol, and ethanol as solvents.

Best results were obtained for FFAs in hexane at�708C and a

10% oil/solvent ratio (Fig. 4) [56]. Use of hexane makes

this procedure suitable when working with alimentary

Table 2. GLA and SDA purities and yields obtained from several FA sources using the urea complexation method

Oil source of FAs

Complexation parameters

Molecular species

SDA% GLA%

Solvent T (8C) Urea:FA ratio (w/w) Puritya) Yieldb) Puritya) Yieldb)

Isochrysis galbana biomass [47] Methanol 4 4:1 FFA 8.5 71.6

Ribes nigrum seeds [48] Methanol 0 5:1 FFA 79.6 11

Anchusa aurea seeds [49] Methanol 0 4:1 FFA 73.2 89.8

Echium fastuosum seeds [49] Methanol 0 4:1 FFA 22.4 63.6 57.3 90.2

Scrophularia sciophila seeds [49] Methanol 2 4:1 FFA 86.2 83.0

Borago officinalis seeds [50] Ethanol �7 3.7:1 FFA 91.5 67.0

Spirulina platensis [51] Methanol 0 10:1 FAME 90.0 64.0

a) GLA or SDA% on total FAs obtained after urea complexation process.b) GLA or SDA% obtained after urea complexation process with respect to the previous FA amount.

Figure 4. Major FA composition ofE. fastuosum seed oils, liquid fraction (LF) composition and yields of each PUFA obtained bywinterization

at S708C from 10, 20, and 40% w/w solutions of seed oil FAs in n-hexane.



purposes, being also useful because it is easy to use at

industrial scale [18].

Fractional crystallization method has been used to obtain

an enriched GLA fraction from borage oil, by a two-stage

process [57]. In the first stage (low-temperature crystalliza-

tion), the solvent employed was acetonitrile (ACN), while in

the second stage the solvent used was a mixture of ACN/

acetone (30:70 v/v). After the first stage, GLA content

increased from 23.4% in borage oil to 66.1% in the purified

fraction with a yield of 93%. After the second stage, GLA

content increased up to 93.9% with a corresponding yield of

92.4% (overall yield 86%). In a posterior report, the same

authors achieved an enriched fraction of 99.1% GLA adding

a third step to the method, a lipase-catalyzed esterification

[58]. However, the use of ACN and acetone together makes

this procedure unsuitable for alimentary applications due to

the risks reported for this mixture [59].

2.3.3 Chromatographic methods

By this procedure, FAs can be separated according to their

carbon number or unsaturation degree by using appropriate

adsorbents [60]. Thus, several chromatographic options are

available to concentrate n-3 and n-6 FAs, which in most cases

are used to obtain EPA and/orDHA:HPLC [47–,49, 49, 61],

silver resin chromatography column [62], silver nitrate-

impregnated silica gel column [63], chromatography column

on silicic acid [64], and cation-exchange Y-zeolite/fixed-bed

column [65]. Until now, GLA has usually been the target FA,

whereas SDA has been scarcely reported when these tech-

niques are used.

The simultaneous purification of SDA and GLA has been

carried out successfully by HPLC. In all cases, a previous

PUFA concentrate is used to purify the desired FA (Table 3).

Notice that starting from E. fastuosum seed oil and after a

three-step process (including oil saponification, FFA obtain-

ing, and PUFA urea concentration) pure fractions of 100%

SDA and 88.6% GLA were obtained after HPLC separation

[49]. Also a purified SDA fraction has been obtained when a

FA concentrate from I. galbana (6.3% SDA) was used as

starting material. Thus, SDA percentage in the urea concen-

trate was 22.6% whereas it reached 94.8% SDA after semi-

preparative HPLC [47].

This technique is readily available when working at ana-

lytical scale. However, it presents several drawbacks related to

the scaling-up when big amount of purified products is

required. Also, it is expensive (solvents, chromatographic

column, staff training. . .) so other alternative must be found

when working at larger scale.

The FAs from E. fastuosum seed oil has also been fractio-

nated by silver ion-silica gel gravimetric chromatography

column, yielding 97.4% GLA ester purity [66]. Also by this

technique, SDA ester has been purified reaching 100% purity

fromEchium plantagineum seed oil FAs (unpublished results).

On the other hand, when this procedure was applied to fish oil

FAs, lower SDA ester purity was achieved because of coe-

lution of other PUFAs in the same chromatographic frac-

tions, although the procedure yielded pure fractions of DHA

[67].

When the aim of oil processing is the further application

with alimentary or pharmaceutical purposes, the election of

proper solvents is essential because only a reduced number of

them (such as n-hexane, acetone or ethanol) are legally

allowed in these types of industries [19]. Thus, by using silver

ion-silica gel gravimetric chromatography column and grade

alimentary solvents, our Research Group has achieved the

purification of both GLA and SDA as enriched TAGs from

some natural sources such as evening primrose (O. biennis)

seed oil (Fig. 5) and viper’s bugloss (E. plantagineum) seed oil

(Fig. 6). Thus, TAGs up to 52.6% GLA on total FAs were

isolated from evening primrose seed oil (10.1% GLA in

original oil) [68] and also TAGs with 30.8% SDA on total

FAs were isolated from viper’s bugloss seed oil (14.0% SDA

in original oil) [69].

GLA ethyl ester has been isolated frommicrobial lipids by

means of a fixed-bed column system where a two-step

desorption operation mode by using zeolites was found to

be effective for selective separation of the ester [65]. Bymeans

of this methodology, GLA ethyl ester (98 mol% purity) was

obtained from a mixture of various PUFAs and unsaturated

Table 3. GLA and SDA purities and yields from several FA sources obtained by HPLC

FA source

HPLC parametersa) SDA% GLA%

Mobile phase

Me:Wab)Flow rate

(mL/min) Puritya) Yieldb) Puritya) Yieldb)

Isochrysis galbana FA urea concentrate [47] 8:2 3 94.8 100

Ribes nigrum FA urea concentrate [48] 9:1 150 95.4 –c)

Anchusa azurea FA urea concentrate [49] 8:2 1.5 94.2 84.6

Echium fastuosum FA urea concentrate [49] 8:2 1.5 100 100 88.6 55.4

a) In all cases a C-18 RP Column was used.b) (w/w) ratios, Me, methanol; Wa, water.c) Not reported.



FA esters using cesium Y and methyl-, dimethyl-, and ethyl-

ammonium Y zeolites. This is the only reference that

describes this methodology with respect to the purification

of any FA ester. Consequently, possible good results for other

FAs remain unexplored until now.

Centrifugal partition chromatography (CPC) is a rela-

tively novel and unexplored technology, which was developed

by Ito et al. [70]. This is a liquid–liquid chromatography

where the sorbent use is obviated, and only two immiscible

solvent phases are needed. This is basically an outgrowth

of countercurrent distribution, as developed by Craig and

Post [71].

Generally in CPC, a liquid phase remains as stationary

while a second solvent phase passes through the solvent that

acts as the stationary phase. The principle of separation

involves the partition of a solute between two immiscible

solvents (mobile and stationary phases). CPC has been

successfully employed for the separation, isolation, concen-

tration, and purification of FAs, phospholipids, and tocopher-

ols compared to traditional liquid–solid separation methods.

CPC does not require the use of solid support as the stationary

phase. Therefore, the possibility of irreversible retention of

highly retentive sample components is eliminated [72].

Few authors described FA purification by this procedure.

Murayama et al. [73] have separated a mixture of ethyl esters

of C18:0, C18:1, C18:2, and C18:3 (GLA) FAs from borago

oil because their partition coefficients are distributed over a

wide range in the two-phase solvent n-hexane/ACN (1:1 v/v).

Figure 5. RP HPLC profile of TAGs from O. biennis seed oil and purified peaks 1 and 2 after chromatographic separation. Comparison

between C18-PUFA content in the original oil and in the purified peaks.



The ethyl esters of C18:2 andGLAwere separated during the

first normal ascending elution whereas C18:1 was recovered

by switching the elution mode. Thus, a final 98.3% GLA

purity was obtained.

Last chromatographic application here exposed is coun-

tercurrent chromatography, coupled with GC-MS for FA

identification, which is applied by Bousquet et al. [74], testing

the separation of SDA, EPA, and DHA from Skeletonema

costatum and I. galbana oils, obtaining purified EPA andDHA

from these oils. The first separation used n-heptane as the

stationary phase and ACN/water (3%) as the mobile phase.

The minor important FAs were removed from these eluted

fractions, leaving a mixture of four major PUFAs. This crude

acid mixture showed better results for SDA isolation in

I. galbana oil, in which this FA reached a 69% on total

FAs in the resulting extract. These authors considered that

this technique may be an alternative to HPLC for the pre-

parative-scale purification of such compounds.

2.3.4 Distillation methods

FAMEs of several mixtures have been partially separated

by this procedure, which takes advantage of differences

in the boiling point and molecular weight of FAs under

reduced pressure [75]. Lower temperatures and short heating

intervals can be employed when molecular distillation and

short-path distillation are used. Fractional distillation is the

most widely used distillation procedure of FAMEs under

reduced pressure (0.1 � 1.0 mm Hg). However, moderately

high temperatures are always required; the more highly unsa-

turated FAs (especially n-3 PUFAs) are, the more prone they

are to oxidation, polymerization, and double bond isomer-

ization. Heated columns packed with glass helices or some

form of metal packing are in common use despite the dis-

advantage of a significant-holdup and pressure drop through

the column [76].

The difficulty of concentrating just n-3 PUFAs from fish

oil in the natural TAG form has been reported [77].

Distillation has been shown to be much more effective by

using FAMEs of FFAs instead of TAGs. Nevertheless, the

method gave reasonable results only for DHA.

In any case, the exposure of long-chain n-3 PUFAs to high

temperatures during distillation may induce FA degradation

by several mechanisms [78, 79]. Therefore, when designing a

method for preparation of n-3 PUFA concentrates; low proc-

ess temperature and time to minimize thermal damage must

be considered.

Distillation method has been used to obtain GLA ester

enriched fractions after lipase-catalyzed modifications of

borage oil due to its ability to separate FFAs from products

Figure 6. RP HPLC profiles of TAGs from E. plantagineum seed oil and SDA-enriched fraction after chromatographic separation.



of lipase-catalyzed reactions [80] (Fig. 7). Concerning to

SDA, this procedure has not been indexed successfully to

obtain SDA concentrates until now.

2.3.5 Enzymatic methods

Lipases are part of the hydrolases family and their physio-

logic role is to hydrolyze TAGs into diacylglycerols, mono-

acylglycerols, and FFAs acting on carboxylic ester bonds [81].

These enzymes are very versatile and in addition to hydrolysis,

they can catalyze different kinds of reactions (Fig. 8), such as

esterification, interesterification, acidolysis, and alcoholysis.

Lipase activity has been known for a long time and many of

them have been widely used for alimentary purposes.

FAs can be esterified, hydrolyzed, or exchanged by lipases

[82]. The reaction is reversible because under low water

activity conditions, the enzyme is able to act ‘‘in reverse,’’

i.e., the synthesis of an ester bond rather than its hydrolysis

[83]. Some variables such as temperature, water concen-

tration, and reaction time influence the direction and effi-

ciency of the reaction [84].

Lipases have been used to discriminate among different

FAs, usually during esterification processes, but also during

hydrolysis. This way, a lipase from Candida cylindracea has

been used to enrich the GLA content up to 46% in the

unhydrolyzed acylglycerols from borage (B. officinalis L.)

and evening primrose (O. biennis L.) oils [85].

To date, lipases have been frequently used to discriminate

between EPA and DHA in n-3 FA concentrates. However,

this procedure has been scarcely used to produce SDA con-

centrates. It is reported that SDA and hexadecatetraenoic

acid (16:4n-3) can be concentrated from some edible marine

algae such asUndaria pinnatifida andUlva pertusa [86]. These

reached up to 40% of total FAs. In order to prepare 16:4n-3

and SDA concentrates, they screened for a suitable lipase to

concentrate them by the removal of other FAs by using a

selective esterification reaction reported by Shimada et al.

[87]. In combination with the lipase reaction and RPmedium

pressure LC, they purified SDA and 16:4n-3 to more than

95% purity [86].

It is expected that new research about SDA concen-

tration/purification by enzymatic procedures can be devel-

oped in a future, considering the potential benefits of

consuming the SDA concentrates in the acylglycerol form.

In contrast with SDA, GLA purification by esterification

processes has been widely reported. Lipases from Pseudomonas

have been found to be suitable for hydrolysis of borage oil [88].

By using this lipase in optimal conditions, the hydrolysis extent

reached 92%, and 93% GLA was recovered as FFA. FAs so

obtained were esterified using Rhizopus delemar lipase increas-

ing GLA content from 22.5 to 70.2 wt%. A second esterifi-

cation reaction was applied to the remaining FFA to obtain a

higher GLA purity. As a result of the whole process, GLA

content in the purified fraction was 93.7 wt% with a recovery

of 67.8% when compared with the initial content.

A process for large scale purification of GLA has been

reported by Shimada et al. [89] by using an oil containing

45% GLA (GLA45 oil) produced by selective hydrolysis of

borage oil as the starting material (Fig. 7). The process

includes a selective hydrolysis by Pseudomonas lipase, two steps

of film distillation, esterification of the distillates using

Rhizopus lipase and lauryl alcohol, isolation of the FFAs frac-

tion after esterification reaction through simple distillation,

and a second esterification process applied to FFAs. After this

last esterification, FFAs recovered by distillation could be

further purified by urea adduct fractionation, obtaining a final

product with a 98.6%GLAcontent and a recovery of 49.4%of

the initial content of GLA45 oil. For industrial application of

this method concerning to GLA purification, the last step

(urea adduct fractionation) could be suppressed if distillation

process is operated under high vacuum.

Distillation

Distillate (GLA45-FFA)

Residue (Glycerides)

Selective esterification with LauOH using R. delemar lipase Distillation

Distillate 1 (LauOH)

Distillate 2 (FFAs)

Residue (Lauryl esters)

Selective esterification with LauOH using R. delemar lipase Distillation

Distillate 1 (LauOH)

Distillate 2 (Purified GLA)

Residue (Lauryl esters)

Hydrolysis with Pseudomonas lipase

GLA45 Oil

Figure 7. Strategy for the large-scale purification of GLA by hydro-

lysis/selective esterification method [89].

Figure 8. Different reactions catalyzed by lipases. Adapted from A.

Houde et al. [81].



Also, interesterification processes have been reported for

GLA enrichment. Spurvey et al. [90] used lipases from

Pseudomonas species and Rhizomucor miehei to incorporate

GLA to seal blubber oil (SBO) and menhaden oil (MO).

Before enzymatic interesterification, the percentage of GLA

in SBO was 0.59% while after the reaction it raised up

to 37.1%. GLA in MO was 0.43% whereas enzymatic

interesterification allowed to increase GLA content up to

39.6%. The advantage of these modified oils is the better

GLA absorption when it is situated at the sn-2 position of

the TAGs.

On the other hand, products with 65% GLA in their

acylglycerol fraction have been achieved employing acidolysis

processes [91] with borage oil as starting material.

Synthesis of structured TAGs containing a specific FA in

sn-2 position has been carried out by using enzymatic alcohol-

ysis to obtain the intermediate sn-2 monoacylglycerol and

further synthesis of the desired TAGs. To date, structured

TAGs containing specific FAs such as EPA, AA, and DHA

[92–94] or palmitic acid [95, 96] have been reported.However,

specific papers on GLA and SDA-containing structured TAGs

synthesis by using alcoholysis have not been reported.

Several processes involving natural sources and a number

of enzymes have been employed to purify GLA in different

forms (FAs, esters, and acylglycerols). Some of the most

representative are shown in this work (Fig. 9) [97–100].

3 Conclusions

Production of pure GLA and SDA from their natural sources

may be achieved using several techniques, specially when

GLA is the target FA. These products may be obtained

mainly as FFA, FAMEs, or TAGs. Due to potential healthy

benefits of the PUFA concentrates in the acylglycerol form,

enzymatic procedures have recently become popular and

their industrial production is a desirable option.

Enzymes are environmentally friendly catalysts that pos-

sess broad substrate specificities, work under mild reaction

conditions, are commercially available and do not require the

use of cofactors. Nevertheless, when obtaining GLA and/or

SDA concentrates, extraction by solubility differences or the

separation of the TAGs containing both FAs from natural oil

sources seem to be the cheapest and most suitable techniques

here reviewed.

In relation to SDA, it is necessary to improve the knowl-

edge about new SDA sources, in which other PUFAs achieve

lower rates. Then, all techniques here reviewed possibly

would offer better results. This is a desirable option, with

the aim of designing experiments that clearly establish the

conversion rate of SDA to EPA.

When purifying both TAGs enriched in GLA and SDA

as well as GLA and SDA methyl esters from their natural

sources such as seed oil from O. biennis and E. plantagineum,

gravimetric normal-phase chromatography with silica gel-

silver nitrate as stationary phase offers suitable results taking

also into account that biocompatible solvents can be used

as mobile phase, allowing the use of these products for

alimentary and/or pharmaceutical purposes. Furthermore

this procedure is simple, easy to scale-up and cheap.

The authors have declared no conflict of interest.

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gamma-linolenic and stearidonic acids: purification and upgrading of c18-pufa oils

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