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Contents lists available at ScienceDirect

Phytochemistry Letters
journal homepage: www.elsevier.com/locate/phytol

Phytochemical characterization of potential nutraceutical ingredients

from Evening Primrose oil (Oenothera biennis L.)
S. Montserrat-de la Paz *, M.A. Fernandez-Arche, M. Angel-Martn, M.D. Garca-Gimenez
Department of Pharmacology, School of Pharmacy, University of Seville, C/ Profesor Garca Gonzalez, 2, 41012 Seville, Spain

A R T I C L E I N F O A B S T R A C T

Article history: Evening Primrose oil (EPO) is a natural product extracted by cold-pressed from Oenothera biennis L.
Received 19 December 2012 seeds. EPO is widely used as a dietary supplement from which benecial effects have been reported in
Received in revised form 17 June 2013 rheumatic and arthritic conditions, atopic dermatitis, psoriasis, premenstrual and menopausal
Accepted 12 August 2013
syndrome, and diabetic neuropathy. The benecial effects of EPO are thought to be due to its g-
Available online xxx
linolenic acid content; in contrast, little effort has been expended to characterize the non-triglyceridic
constituents of EPO. In order to evaluate its potential as source of functional food ingredients our aim in
Keywords:
this work has been identied and quantied the different components of EPO by different techniques
Functional food
Unsaponiable
(GCMS and HPLC). The lipid prole showed that oleic (7%), linoleic (74%) and g-linolenic (9%) were the
Oil most abundance fatty acids. Unsaponiable matter and subfractions were obtained by CEE/2568/91.
Phytosterols Separation of the compounds under study was achieved giving a reasonable analysis time and good
Policosanol resolution. A yield (1.821.95%) of unsaponiable matter was obtained and levels of saturated
Evening Primrose hydrocarbons (0.291.97  14.85 mg) were noticed. b-Sitosterol (7952.00  342.25 mg/kg oil) and
campesterol (883.32  0.45 mg/kg oil) were predominant in phytosterol fraction (9573 mg/kg oil), while
tetracosanol (236.93  2.32 mg/kg oil) and hexacosanol (289.92  3.41 mg/kg oil) in linear aliphatic alcohol
fraction (798.04  5.66 mg/kg oil). In the phenolic fraction (55.49  2.76 mg/kg oil), ferulic acid
(25.23  2.64 mg/kg oil) was the major component. From the results obtained, it can be suggested that
the Evening Primrose oil can be considered an interesting alimentary source of substances of nutraceutical
value.
2013 Phytochemical Society of Europe. Published by Elsevier B.V. All rights reserved.

1. Introduction portion, about 1.52% of the oil, contains elevated amounts as

Oil obtained from the seeds of Evening Primrose (Oenothera
biennis L., Onagraceae) has attracted much interest due to its high
content of polyunsaturated fatty acids, in particular, g-linolenic
acid (18:3n-6) (Hudson, 1984). Evening Primrose oil (EPO) is
widely used as a dietary supplement from which benecial effects
have been reported in rheumatic and arthritic conditions, atopic
dermatitis, psoriasis, premenstrual and menopausal syndrome,
and diabetic neuropathy (Mahady et al., 2001). However, the
present clinical evidence for most uses needs to be substantiated
by further rigorous trials (Belch and Hill, 2000; Kleijnen, 1994). The
benecial effects of EPO are thought to be due to its g-linolenic acid
content, since v-6 fatty acids are precursors for eicosanoids of the
1-series and exert an inhibitory effect on leucotriene synthesis
(Belch and Hill, 2000).
In contrast, little effort has been expended to characterize the
non-triglyceridic constituents of EPO. The non-saponiable
* Corresponding author. Tel.: +34 605383541.
E-mail address: delapaz@us.es (S. Montserrat-de la Paz).
sterols, 4-methyl sterols, tocopherols, triterpene alcohols, hydro-
carbons and alcohols (Hudson, 1984). As part of ongoing
investigations on biactive secondary plant metabolites in medici-
nal and food plants (Fernandez-Arche et al., 2009), we recently
carried out some preliminary analytical screening of EPO
(Montserrat-de la Paz et al., 2012). Therefore, the present study
was aimed at analyzing the potential nutraceutical ingredients of
EPO.
2. Results and discussion
Evening Primrose oil (EPO) is best known for its use as a dietary
supplement which benecial effects have been reported. The lipid
prole from EPO showed that oleic (7%), linoleic (74%) and g-
linolenic (9%) were the most abundance fatty acids (Fig. 1 and
Table 1) and its therapeutic effects are attributed to the direct
action of its essential fatty acids (Fan and Chapkin, 1998). The role
of n-6 is not fully elucidated yet, as g-linolenic and dihomo-g-
linolenic acids are precursors of the 1-series prostaglandins, which
have anti-inammatory properties, the observed increase of these
fatty acids can be regarded as a favorable effect. However, little

1874-3900/$ see front matter 2013 Phytochemical Society of Europe. Published by Elsevier B.V. All rights reserved.
http://dx.doi.org/10.1016/j.phytol.2013.08.008

Please cite this article in press as: Montserrat-de la Paz, S., et al., Phytochemical characterization of potential nutraceutical ingredients
from Evening Primrose oil (Oenothera biennis L.). Phytochem. Lett. (2013), http://dx.doi.org/10.1016/j.phytol.2013.08.008
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Table 1 Table 2
Fatty acid prole (GC) in EPO. Amount of 4-desmethylsterols, erythrodiol and uvaol (GC) in EPO.

Peak Retention time Name %  S.E.M. Peak Retention time Name mg/kg oil  S.E.M.

1 8.240 Palmitic acid (16:0) 6.31  0.14 1 9.61 a-Colestanol (I.S.)

2 11.873 Estearico acid (18:0) 1.88  0.02 2 11.88 Campesterol 883.32  0.45
3 13.171 Oleic acid (18:1; v-9) 6.93  0.02 3 13.94 Clerosterol 120.44 0.12
4 13.337 Vaccenic acid (18:1, v-7) 0.81  0.03 4 14.60 b-Sitosterol 7952.00  342.25
5 15.457 Linoleic acid (18:2, v-6) 73.88  0.09 5 14.74 Sitostanol 167.01  39.77
6 16.663 Eicosanoic acid (20:0) 0.31  0.03 6 15.02 D5-Avenasterol 429.65  75.20
7 16.902 g-Linolenic acid (18:3, v-6) 9.24  0.05 7 15.87 D5-24-Estigmastadienol 94.60  5.68
8 17.847 Eicosenoic acid (20:1, v-9) 0.55  0.01 8 16.34 D7-Estigmasterol 38.17  14.33
9 21.911 Docosanoic acid (22:0) 0.10  0.01 9 17.00 D7-Avenasterol 27.80  16.07

Total sterols 9573.24  180.07

10 21.68 Erythrodiol 23.73  1.92
effort has been expended to study other minor compounds present 11 23.03 Uvaol 16.94  4.50
in EPO. In the present work has been isolated and characterized the
different and interesting fractions from EPO. A great amount of
qualitative information was generated in every analysis. A yield reduce biomarkers of oxidative stress and inammation. Plant
(1.821.95%) of unsaponiable matter was obtained. sterols and stanols exert their hypocholesterolemic effects
Sterols were isolated from the EPO unsaponiable matter and possibly by interfering with the uptake of both dietary and biliary
represented a 53.16% from this fraction and almost a 1% from EPO. cholesterol from the intestinal tract (Devaraj and Jialal, 2006).
This high proportion shows that EPO is one of the richest naturals The identication of the different phytosterols have been made
products in phytosterols versus others vegetal oils frequently used by GC and GCMS comparing their RRt values and their MS
in the diet like from corn (0.95%) (Ostlund et al., 2002) or from fragments with literature values. Chromatographic analyses are
sunower (0.73%), otherwise the famous olive oil only have a 0.17% shown in Fig. 2 and Table 2, b-sitosterol (7952.00  342.25 mg/kg
of phytosterols (Weihrauch and Gardner, 1978). Plant sterols and oil) and campesterol (883.32  0.45 mg/kg oil) were predominant in
stanols in fat matrices effectively lower LDL cholesterol levels in phytosterol fraction (9573 mg/kg oil).
hypercholesterolemic, diabetic, and healthy human volunteers. Another interesting fraction is the linear aliphatic alcohols.
Recent studies also show that sterols (2 g/d) lower LDL cholesterol Tetracosanol (236.93  2.32 mg/kg oil) and hexacosanol
even when incorporated in nonfat matrices. In addition, they may (289.92  3.41 mg/kg oil) were predominant in linear aliphatic

Fig. 1. Gas chromatogram of fatty acid prole from Evening Primrose oil.

Fig. 2. Gas chromatogram of the 4-desmethylsterols, erythrodiol and uvaol fraction from Evening Primrose oil.

Please cite this article in press as: Montserrat-de la Paz, S., et al., Phytochemical characterization of potential nutraceutical ingredients
from Evening Primrose oil (Oenothera biennis L.). Phytochem. Lett. (2013), http://dx.doi.org/10.1016/j.phytol.2013.08.008
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S. Montserrat-de la Paz et al. / Phytochemistry Letters xxx (2013) xxxxxx 3

Fig. 3. Gas chromatogram of linear aliphatic alcohols, triterpene alcohols and 4-methylsterols fraction from Evening Primrose oil.

Fig. 4. Gas chromatogram of phenols from Evening Primrose.

alcohol fraction (798.04  5.66 mg/kg oil) (Fig. 3 and Table 3). 2006). On the other hand, in recent investigations performed by our
Chemical and nutritional studies have shown that mixtures of long- group demonstrated that similar linear alcohols fraction isolated from
chain primary alcohols extracted from waxy materials from different pomace olive oil (a by-product of olive oil) have a protective effect on
sources, such as beeswax, rice bran, wheat germ, sugar cane and grain some mediators involved in the inammatory damage development
sorghum, are able to exert several benecial physiological effects, (Fernandez-Arche et al., 2009).
such as reducing platelet aggregation, endothelial damage and Table 4 proved to be not rich in squalene, which presence was
cholesterol-lowering effects (Arruzazabala et al., 2000; Singh et al., 0.40 mg/kg from EPO if compared with other known plant rich in
squalene (olive oil or Amaranthus oil) (Ostlund et al., 2002). The
Table 3 levels of saturated hydrocarbons were of 291.97  14.85 mg
Amount of linear aliphatic alcohols, triterpene alcohols and 4-methylsterols (GC) in Phenolic compounds make important contributions to the
EPO. nutritional properties and sensory characteristics. These com-
Peak Retention time Name mg/kg oil  S.E.M.
pounds play an important role in human health because of their
anti-inammatory, anti-allergic, antimicrobial, anti-carcinogenic
Linear aliphatic alcohols
and antiviral activities (Medina et al., 2007) The phenolic content
1 2.28 Phytol 29.06  0.12
2 2.87 Geraniol 18.94  0.09 of EPO (55.49  2.76 mg/kg oil) was reported in Fig. 4 and Table 5 and
3 3.84 C21 (I.S.) ferulic acid (25.23  2.64 mg/kg oil) was the major component.
4 4.67 C22 85.24  0.42 Ferulic acid can be found in whole grains include whole grain cereals,
5 5.66 C23 18.80  0.06
whole grain breads, brown rice and rolled oats. According to Third
6 6.82 C24 236.93  2.32
7 8.10 C25 33.87  0.18
Planet Food, the highest concentrations of ferulic acid are in the bran
8 9.53 C26 289.92  3.41 of these grains. This is a good reason why choosing whole grain foods
9 11.04 C27 27.97  0.05
10 12.56 C28 57.31  0.18
Table 4
Total policosanols 798.04  5.66
Amount of squalene, linear hydrocarbons and waxes (GC) in EPO.
Triterpene alcohols
Peak Retention time Name mg/kg oil  S.E.M.
11 16.05 Dammaradienol 281.71  5.32
12 16.50 Taraxterol 414.64  4.36 Total linear hydrocarbons 291.97  14.85
13 17.29 b-Amirin 996.51  8.09 1 6.458 Lauryl Arachidate (I.S. 1)
14 17.65 Butirospermol 69.66  1.02 2 10.808 Squalene 0.40  0.05
15 18.19 Cycloartenol 81.93  2.11 3 13.943 Methyl heptadecanoate (I.S. 2)
16 19.07 24-Methylencycloartenol 210.07  4.65 Aliphatic waxes 1.26  0.12
Terpenic waxes 40.35  0.31
4-Methylsterols
17 20.73 Citrostadienol 392.60  4.57 Total waxes 41.61  0.41

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Table 5 three times with 80 mL of ethyl ether. The ether extracts were
Amount of phenols (HPLC) in EPO.
pooled into a separating funnel (with 125 mL of distilled water)
Peak Retention Name mg/kg oil  S.E.M. and washed with distilled water (125 mL each time), until the
time wash gave a neutral reaction. Then the wash water was removed,
1 10.699 Hydroxytyrosol 1.11  0.05 and the organic sample was dried with anhydrous sodium sulfate,
2 14.357 4-hydroxylphenolacetic acid (I.S. 1) ltered, taken to dryness and the residue was weighed.
3 16.431 Vanillic acid 3.27  0.63
4 19.304 Vanillin 17.37 1.03
3.4. Separation of the unsaponiable components: sterol, linear
5 22.778 p-Cumaric acid 1.75  0.01
6 24.168 Pherulic acid 25.23  2.64 aliphatic and triterpen alcohol and 4-methylsterol fractions
7 28.001 o-Cumaric (I.S. 2)
8 29.152 1st derivate hydroxytyrosol 6.76  0.81 EPO unsaponiable matter was by means of both ash and
Total phenols 55.49  2.76 plate chromatography, providing fractions with superimposable
proles. Preparative TLC was preferred over column chromatog-
raphy due to its greater handiness and convenience of operation.
instead of rened ours and breads are a healthier choice. This Unsaponiable fraction dissolved in chroloform was deposed on a
phytonutrient is a popular sports supplement for its ability to 1 mm silica gel 60 F254 PLC Plate 20  20 (Merck, Darmstadt,
neutralize free radicals in muscle tissue which can cause muscle Germany). The plate was placed into the developing chamber and
fatigue, loss of endurance, decreased performance, and muscle eluted with hexanediethylether (65:35, v/v), after developing
soreness. It is also marketed as a remedy for hot ashes, and other and solvent evaporation, the plate was developed with 20 ,70 -
health ailments that can be caused by inammation and oxidation of dichlorouorescein solution and important bands was evidenced.
tissues (Kim et al., 2003). Coffee, amaranth, artichokes, peanuts, The most important bands were scraped separately from the plate,
oranges, pineapple and apples are also good sources. It is usually and extracted rst with hot chloroform (10 mL) and then with
found in the cell wall, but also in the seeds and leaves of a plant. ethyl ether (10 mL). The two solvent extracts were mixed and
taken to dryness. The residue was nally dissolved and sylilated
3. Materials and methods with 200 mL of a mixture of 9:3:1 v/v/v of pyridinehexamethyl-
disilazanetrimethylchlorosilane to 1530 mg of the insaponic-
3.1. Standards and reagents ables in glass-stoppered tubes (Cert et al., 1997).

Standards of a-cholestanol, eicosanol, methyl heptadecanoate 3.4.1. Chromatographic conditions

and lauryl arachinodate (the purity of all standards were greater
than 95%) and the rest reagents were purchased from Sigma
Aldrich Chem. (St. Louis, MO, USA).
3.2. Determination of fatty acids prole
3.2.1. Sample preparation
The fatty acid composition was determinated by gas chroma-
tography following the methylation with cold methanolic solution
of potassium hydroxide. In a 5-mL screw top test tube, weigh 0.10 g
of the EPO sample. Add 3 mL of heptane and 500 mL of 2 N
methanolic potassium hydroxide solution. Shake vigorously for
15 s. Leave to stratify until the upper solution becomes clear.
Decant the upper layer containing the methyl esters (Cert et al.,
2000a).
3.2.2. Chromatographic conditions
The fatty acid methyl esters were analyzed in a Hewlett-
Packard 6890 gas chromatograph equipped with ame ionization
detector, using a SP2380 silica capillary column (30 m  0.32 mm
internal diameter, 0.25 mm lm). The initial column temperature
was 165 8C, which was held for 10 min, the programmed from
165 8C to 200 8C at 1.5 8C/min. Injector temperature was 210 8C
and detector temperature was 250 8C.
3.3. Plant material and unsaponiable extraction
EPO was obtained from MARNYS1 (Cartagena, Spain). The
unsaponiable matter of EPO was isolated following conventional
procedures and its components were analyzed following the IUPAC
method (Paquot, 1992). 5 g of Evening Primrose seed oil, 1 mL of
internal standard solution (a-colestanol 1 mg/mL and eicosanol
0.5 mg/mL in chroloform) and 50 mL of a 2 N potassium hydroxide
solution in ethanolwater (80:20, v/v) were put into a 250 mL
ask; saponication was carried out by boiling and stirring the
sample for 1 h. After cooling, 100 mL of distilled water were added,
and the sample transferred to a separating funnel and extracted
Sterols and alcohols fractions were analyzed with a HP5890
Series II gas chromatograph (Boston, USA) equipped with a
capillary column SPB5, 30 m length, 0.25 mm i.d. and 0.25 mm
lm thickness of poly(5% diphenyl/95% dimethyl siloxane)
stationary phase. Carrier gas was hydrogen, with a head pressure
of 110 kPa and 1:60 split ratio. Injector and detector temperature
were 300 8C. Isotherm program to 255 8C was programmed to
sterols fraction during 30 min and the oven temperature was
programmed from 225 8C to 280 8C with a rate of 3 8C/min for
alcohols fraction for 50 min The analysis was carried out following
the amending Regulation (EC) 2568/91 on the characteristics of
olive and the relevant method of analysis. The MS analyses were
performed using Kratos MS 80 mass spectrometer equipped with a
NBSLIB2 data system. Fraction components were quantied by
means of the internal standards and total concentrations in EPO
was calculated as the sum of individual peak concentrations.
3.5. Determination of hydrocarbon and wax contents
The wax esters, formed by the reaction of alcohols (aliphatic,
triterpenic, methylsterols and sterols) and free fatty acids are
present in seed and fruits. The usual procedure was followed by
column chromatographic. This method has been adopted by
European Union. The separation is affected by the composition of
the mobile phase (hexane:diethyl ether, 99:1), the temperature
and the activation of the silica gel. A solution of lauryl
arachinodate (0.1 mg/mL) and methyl heptadecanoate (0.1 mg/
mL) in methanol was used as internal standard. The wax esters
eluted just before the triacylglycerols, thereby, the addition of a
colorant (Sudan I) with Rf similar to that of the triacylglycerols
allows to visualize when the elution of wax esters ends. The
fraction isolated contains also other minor components such as
the hydrocarbons. After isolation, this fraction was carried out by
gas chromatographic system (Agilent Technologies 6890N)
equipped with on-column injecto, FID and using a short capillary
fused-silica column (10 m) coated with 5% phenylmethylsilicone
(Cert et al., 2000b).

Please cite this article in press as: Montserrat-de la Paz, S., et al., Phytochemical characterization of potential nutraceutical ingredients
from Evening Primrose oil (Oenothera biennis L.). Phytochem. Lett. (2013), http://dx.doi.org/10.1016/j.phytol.2013.08.008
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3.6. Analysis of phenolic compounds
3.6.1. Internal standard and sample preparation
A solution of p-hydroxyphenylacetic (4.64  102 mg/mL) and
o-coumaric acids (9.6  103 mg/mL) in methanol was used as
internal standard. A sample of EPO (2.5  0.001 g) was weighed, and
0.5 mL of standard solution was added. The solvent was evaporated in
a rotary evaporator at 40 8C under vacuum, and the oily residue was
dissolved in 6 mL of hexane.
3.6.2. SPE
A diol-bonded phase cartridge from ISCO1 was placed in a
vacuum elution apparatus and conditioned by the consecutive
passing of 6 mL of methanol and 6 mL of hexane. The vacuum was
then released to prevent drying of the column. The oil solution was
applied to the column, and the solvent was pulled through, leaving
the sample and the standard on the solid phase. The sample
container was washed with two 3-mL portions of hexane, which
were run out of the cartridge. The sample container was washed
again with 4 mL of hexane/ethyl acetate (90:10, v/v), which were
run out of the cartridge and discarded. Finally, the column was
eluted with 10 mL of methanol, and the solvent was evaporated in
a rotary evaporator at room temperature under vacuum until
dryness. The residue was extracted with 500 mL of methanol/water
(1:1, v/v) at 40 8C. An aliquot (20 mL) of the nal colorless solution
was injected into the HPLC system.
3.6.3. High-performance liquid-chromatography
HPLC was performed in a Hewlett-Packard series 1100 liquid
chromatographic system equipped with a diode array UV detector
and a Rheodyne injection valve (20-L loop). A Lichrospher 100RP-
18 column (4.0 mm i.d.  250 mm; particle size = 5 mm) (Merck),
maintained at 30 8C, was used. Elution was performed at a ow rate
of 1.0 mL/min, using as mobile phase a mixture of water/acetic acid
(97:3, v/v) (solvent A) and methanol/acetonitrile (50:50, v/v)
(solvent B). The solvent gradient changed according to the
following conditions: from 95% (A)5% (B) to 70% (A)30% (B) in
25 min; 65% (A)35% (B) in 10 min; 60% (A)40% (B) in 5 min; 30%
(A)70% (B) in 10 min; and 100% (B) in 5 min, followed by 5 min of
maintenance. Chromatograms were acquired at 240, 280, and
335 nm (Mateos et al., 2001).
3.7. Statistical analysis
The mean response factors of all the classes of analytes (sterols,
alcohols, hydrocarbons, waxes, fatty acids, phenols and tocopher-
ols) were calculated respect to respective internal standards. Each
data collected comes from the average of three determinations and
the standard error (S.E.M.) is given. Each chromatogram is the most
representative of three similar analyses.
4. Conclusions
The results obtained in this study show that interesting
phytochemical compounds are present in Evening Primrose oil.
Some of them have important pharmacological activities. There-
fore we conclude that Evening Primrose oil is an important source
of bioactive compounds with signicant nutritional value.
Please cite this article in press as: Montserrat-de la Paz, S., et al., Phytochemical characterization of potential nutraceutical ingredients
from Evening Primrose oil (Oenothera biennis L.). Phytochem. Lett. (2013), http://dx.doi.org/10.1016/j.phytol.2013.08.008
5
Disclaimer
All authors participated in protocol development, conception
and design of the study, result evaluation, writing, editing and have
approved the nal version of the manuscript. None of the authors
had any nancial or personal interest in any company or
organization sponsoring the research.
Conict of interest
This work does not present conicts of interest.
Acknowledgements
We are grateful to Dr. Carmen Perez-Camino (Department of
Food Characterization and Analysis, Spanish National Research
Council) who collaborated in the identied components in this
study.
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