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Determination of the fatty acids and oil content of evening primrose (Oenothera biennis L.)
Food Reseurch International
26 (1993) 181-186
Determination of the fatty acids and oil content of evening primrose (Oenothera biennis IL.) William A. Court, John G. Hendel Chemistry
Luhoratory.
Agriculture
Canada Research Station,
& Robert Pots
PO Bos 186. Delhi, Ontario, Cunada N4B 2 WY. *
A rapid method for the analyses of oil content and individual fatty acids of evening primrose (Oenothera hiennis L.) is described. Oil was extracted from the seed with a Polytron probe generator using petroleum ether. A portion of the extract was concentrated to determine oil content. Results obtained with the Polytron extraction procedure were comparable to those obtained with a Soxhlet extraction procedure using the same solvent. Saponification of the oil with methanolic sodium hydroxide, methylation with methanolic sulfuric acid, and extraction of the esters with hexane was accomplished in a single reaction vial. Quantitation of the extracted esters was carried out by capillary gas chromatography. A number of fatty acids were identified by gas chromatographic-mass spectrometric studies of the extracted esters and methyl esters separated by their degree of unsaturation with silver nitrate-silicic acid chromatography. Saturated fatty acids from C,, to Cz4 were present in the oil with the even numbered acids present in the larger amounts. Eight monounsaturated fatty acids from C14,, to C,,: , were identified with C,, , (n-9) as the major constituent of this fraction. Seven polyunsaturated fatty acids were identified with C,,:, (n-6) and C,,:, (n-6) as the major component. Analysis of seeds from different parts of evening primrose plants showed that differences in gamma-linolenic [C,, : (n-6)] acid and oil content exist due to location of the seed pods. Keywords:
Oenothera
gas chromatography,
biennis, oil, fatty acids, gamma-linolenic gas chromatography-mass spectrometry.
INTRODUCTION
large amounts of gamma-linolenic acid (Baker, 1987). Analyses of individual plant selections for oil content and individual fatty acids requires a method that minimises seed utilisation; however, an unexpected problem encountered by local producers was unreliable and unreproducible analyses of individual plant selections and commercial crops for gamma-linolenic acid and oil content which has been previously reported (Hudson, 1984). In addition, differences have been reported in the minor fatty acids present in evening primrose seed (Lotti et al., 1984; Bottazzi et al., 1985; Muderhwa et al., 1987; Singer et al., 1990, Gibson et al., 1992). Since it is possible that these differences are due to genetic differences, an examination of the minor fatty acid components of these local plant types would be useful. The object of the study was to develop a fast and accurate method for the determination of oil and individual fatty acid content of evening primrose seed which minimises seed utilisation, and
Evening primrose (Oenothera biennia L.) is a common weed that is native to North America. The species is drought tolerant and grows well on sandy soils (Hall et al., 1988). Evening primrose is of interest because the seeds contain an oil that is rich in gamma-linolenic acid. Although this essential fatty acid has been found in other oilseeds, evening primrose is the most common commercial source (Wolf et al., 1983). Evening primrose is cultivated in a number of countries and because of possible nutritional and pharmaceutical applications, the demand for the oil continues to grow (Carter, 1988). Evening primrose is produced in Ontario, Canada using biotypes selected from local wild populations because these plant selections contain *Contribution
acid, capillary
number 223.
Food Research International 0963-9969/93/$06.00 0 1993 Canadian Institute of Food Science and Technology 181
which is capable of handling the large sample numbers generated by production and plant breeding research programs. In addition the study was conducted to identify the fatty acids present in the individual plant selections found in the region which are used for commercial production.
imately 5 min with dry nitrogen. ‘The weight IIIcrease in the vial was used to calculate the pcrcentage of oil extracted. Corrections wcrc made for the amount of solvent. the internal standard. and the volume change due to the oil extracted. Fatty acid analysis
MATERIALS AND METHODS Reagents and materials
Standard fatty acids and methyl esters were obtained from Sigma Chemical Co., St Louis, MO, Supelco Canada Ltd, Oakville, ON, and Mandel Scientific Co., Guelph, ON. Solvents were obtained from Caledon Laboratories, Georgetown, ON. Except for methanol, which was dried with magnesium, solvents were glass distilled before use. Except for silver nitrate which was obtained from Fisher Scientific Toronto, ON, all other reagents were obtained from J.T. Baker Chem Co., Toronto, ON. Silicic acid (BIO-SIL A) was obtained from Bio-Rad Laboratories, Mississauga, ON. Evening primrose, borage and canola seed were grown at the Agriculture Canada Research Station, Delhi, ON. All seed samples were hand cleaned prior to extraction. Moisture determination
The moisture content of evening primrose seed was determined by heating a sample in a convection oven for 3 h at 130°C. Oil extraction
A 250 mg sample of evening primrose seed was transferred to a tared 25 ml scintillation vial. Ten millilitres of petroleum ether (boiling point 40-60°C) containing the internal standard (tridecanoic acid) were added. The mixture was homogenised for 3 min with a Polytron PTl OST (Brinkmann Inc., Rexdale, ON) probe generator. Periodically during the extraction and after completion the sides of the vial were washed with small portions of petroleum ether. In addition, the probe generator was thoroughly rinsed with ether after homogenisation. After petroleum gently mixing, the vial was capped and the solid material allowed to settle. A 7 ml portion of the micella was then transferred to a tared 3 g sample vial. The solvent was allowed to evaporate in a fume hood with the last traces removed in approx-
Fatty acids were determined by saponifying the oil in the 3 dram sample vial with 2 ml of 0.5 N NaOH in dry methanol at 70°C for 40 min (Court et al.. 1984). After cooling. 1.5 ml of .5’::1H,SO, in dry methanol were added and the mixture reheated at 70°C for 30 min. After cooling, 2.5 ml of saturated NaCl and 4 ml of hexane were added. The vial was shaken for 2 min and the phases allowed to separate. A portion of the hexane layer was rcmoved and analyzed by gas chromatography. Silicic acid-silver nitrate chromatography
Fatty acid methyl ester mixtures were separated according to the degree of unsaturation with 10% silver nitrate on silicic acid according to De Vries (1963). The separation of 650 mg of esters was carried out with 65 g of absorbent. The elution of the methyl esters was with benzene/petroleum ether/ether mixtures (De Vries, 1963; Craig & Bhatty, 1964) or hexane/ether mixtures (Willner, 1965). Gas chromatography Quadrex C’PS- I
Quantitative analyses were performed on a Hewlett-Packard 5890 gas chromatograph equipped with dual FID detectors. A Quadrex CPS- 1 (Quadrex Corp., New Haven. CT) fused silica capillary column (25 m X 0.25 mm i.d., film thickness of 0.25 pm) was employed. One-microlitre injections were made with a HewlettPackard 7672A autosampler. The column oven was temperature programmed from 120 to 200°C at 3”Umin and held to 200°C for 4 min. A slightly longer final time was required if the CZli methyl ester was to be determined. The injector temperature was 183°C detector 200°C. and helium flow was 16.9 cm/s. The split (1 : 45) injection mode was utilised. Supelco
Supelcowllx
10
Analyses were performed in a Hewlett-Packard 5840 gas chromatograph equipped with dual FID detectors. A Supelco Supelcowax 10 (Supelco Canada
Fatty acid and oil content qf evening primrose
183
Ltd, Oakville, ON) fused silica capillary column (60 m X 0.25 mm i.d., film thickness 0.25 pm) was employed. The column oven was temperature programmed from 190 to 240°C at 2”C/min and held at 240°C for 35 min. The injector temperature was 225°C detector 225°C and helium flow was 16.7 cm/s. The split (1 : 50) injection mode was utilised.
Table 1. Comparison of procedures for the determination of oil content of evening primrose
Gas chromatography/mass spectrometry
i
GC/MS analyses were performed on a HewlettPackard 5971 MSD system equipped with a Quadrex CPS-1 (25 m X 0.18 mm i.d.) fused silica capillary column (film thickness 0.25 pm). Onemicrolitre injections were made with a HewlettPackard 7673A autosampler. The splitless injection mode was utilised. The column oven was temperature programmed as follows: 120°C for 1 min, 3”/min to 190°C and held at 190°C for 45 min. The injector temperature was 185°C and helium flow 16 cm/s. Sample components were tentatively identified by mass spectra matching with a Wiley Mass Spectra data base (McLafferty & Stauger, 1989). Tentative identifications were verified by comparison of retention times and mass spectra with that of authentic reference standards where possible, or by previously characterised mixtures such as borage (Wretensjo et ul., 1990) and canola (Ackman, 1986).
Sample N0
Soxhlet extraction
Polytron
extraction
Mean” (‘%,)
SDh
CV’ (‘XI)
Mean” (‘X,)
SD
CV (‘%I)
26.5’ 26.3 24.3
0.18 0.08 0.10
o-7 0.3 0.4
26.7 ‘6-7 74.5
0,41 0.41 0.32
I.5 I.5 l-3
1 3
~~. _ .~.
~~~~~
“Mean value of six determinations. ‘Standard deviation. ‘Coefficient of variation. “Mean value of seven determinations. Values reported are in percentage on a dry weight basis.
tive standard deviation from the mean for the Polytron extraction procedure was greater than that for the Soxhlet procedure but was still quite acceptable. For routine analyses 250 mg of seed were used; however. this was not a limiting factor and smaller seed samples can be readily accommodated. Fatty acid methyl esters were separated and quantitated by capillary gas chromatography. Reproducibility studies for individual fatty acids with two representative evening primrose samples are given in Table 2. Seven individual samples were analysed to obtain this data. Except for several of the very minor fatty acids, acceptable reproducibility was obtained.
Influence of seed pod position
Table 2. Reproducibility data for replicate analyses of evening primrose fatty acids by gas chromatography
Plots were grown at the Agriculture Canada Research Station, Delhi, ON. Mature plants were hand harvested and divided into eight parts as shown in Table 4. Ten plants were harvested and combined to yield each sample. Two replications were employed for each sample.
Acid
RESULTS AND DISCUSSION A number of methods have been used for the determination of the oil content of evening primrose (Lotti rt ul., 1980; Bottazzi et al., 1985; Yaniv et al., 1989). Among these methods the Soxhlet extraction of ground seed has been used extensively. In order to evaluate the effectiveness of the Polytron extraction procedure for the determination of the oil content of evening primrose, it was compared to the Soxhlet procedure using petroleum ether as the extraction solvent (Table 1). Agreement between the two procedures was quite good; however, the rela-
Sample
c I6 C,, c‘,, C,: C,; C,, CP Cl, C,x C,, CIK C?,, C?,,
I (n-9)
i (n-7) / (n-8) I (n-9) I (n-7) I (n-6) i (n-6) 1 (n-3)
I (n-9) Czri 2(n-6) CZ CZl
1”
Sample 2”
“/;,h
CV” (%I)
// b
0.04 0.07 0.03 6.44 0.04 0.07 0.06 0.04 1.81 9.76 0.84 71.20 8.72 0.18 0.31 0.23 0.03 0.12 0.04
7.1 4.8 7 I 1.4 7.2 9.1 9.1 6.2 06 0.8 1.5 0.1 0.3 39 9-5
0.03 0.07 0.03 5.93 0.04 O-04 0.07 O-02 2.08 4.99 0.56 74.40
104 3.9 IO.0 46
‘lMean value of seven determinations. ‘Weight per cent of total fatty acids. ‘Coefficient of variation.
IO.80 0.17 o-31 0.23 0.05 0.13 O-06
cv (0%) 7.5 12-3 74 0.4 8.3 17.8 8.8 3-6 I.6 I.1 2-3 0.2 0.1 7.6 4.5 5.2 5.0 11-3 7.5
Table 3. Fatty acids of evening primrose (Oenotheva hiennis L.) wild biotypes Peak No.
Fatty acid
Saturated
I
c‘12 c’,J C,, C,, Clh C,T C,, C,, Czcr
Identification
SP-IO”
0.403 0,495 O-556 0,664 0,776 0.888
0.419 0.495 0.550 0,639 0.751 0,864 I~000 1.142 I.313 I.515 I.766 2.076 2.468
GC-MS’, X-MS, CC-MS, GC-MS. GC-MS, GC-MS, GC-MS. GC-MS, GC-MS. GC-MS, GC-MS. GC-MS, GC-MS,
RT” RT RT RT RT RT RT RT RT RT RT RT RT
0.567 0,658 0.773 0.780 0,899 I.035 I.358
GC-MS GC-MS GC-MS. GC-MS. GC-MS, GC-MS, GC-MS. CC-MS.
RT RT RT RT RT RT
0.972 I .086 I.115 I.158 I-207 I-264 I.455
GC-MS GC-MS GC-MS. GC-MS, GC-MS, GC-MS. GC-MS.
RT RT RT RT RT
I.109 I.215 I.357 I .442 I -629 I.774
C2l
fatty acids
C 14 I C I? I ;I6 , (n-9) Ih. I Crj I Cl*. I C,,, I c 2. ,
Polyunsaturated I2 I6 17 I8 20 21 25
time
C‘PS-I”
1.000
Czz CY c 24
Monosaturated 4 6 8 9 II 14 I5 24
retention
fatty acids
;7 5 I IO 13 19 23 26 27 28 29
Relative
(n-7) (n-8) (n-9) (n-7) (n-9)
0.564 0.670 0.793 0.802 0.910 1.021 I.029 I.234
I.042
fatty acids
c,,,, C,, 2 CIx.2 (n-6) C,,, (n-6) C,# 3 (n-3) C18.4 (n-3) C 2. 2 (n-6)
0.966 I.052
I.080 I.101 I.131 1.159 I.283
“Quadrex CPS-I capillary column (25 m X 0.35 mm i.d.). “Supelco Supelcowax IO capillary column (60 m X 0.25 mm i.d.). ‘Gas chromatography-mass spectroscopy. “Retention time.
In order to facilitate identification of the minor fatty acids present in evening primrose oil, the methyl esters were separated according to their degree of unsaturation by column chromatography on a silver nitrate-silicic acid absorbent (De Vries, 1963). Saturated fatty acids from C,, to Cl4 were present in the column fractions with the even numbered acids present in larger amounts than the odd numbered fatty acids (Table 3). Except for the C,,, CIy, C2,, and Cl3 acids, all the other saturated fatty acids have been reported previously in evening primrose (Lotti et al., 1980, 1984). A large number of monounsaturated fatty acids have been reported in evening primrose oil with various studies reporting different components
(Lotti et d., 1980; Bottazzi et LII., 1985: Gibson c’l ui., 1992). Examination of the oil samples and unriched column fractions by gas chromatography indicated the presence of a number of monounsaturated fatty acids with substantial amounts of eight monounsaturated fatty acids (Table 3). The major component of this fraction was C !, (11-9) fatty acid with C,, , and C,, , as very minor components which were only evident in the enriched column fractions. Bottazzi et al. t 1985) reported the presence of shorter monoene fatty acids under certain conditions: however, if present they were in very small amounts in the samples examined in this study. Lotti et rrl. (1980) reported that the fatty acid content of evening primrose oil was dependent on the ripeness of the seeds. In order to examine this possibility, seeds were collected IO weeks prior to harvest and their fatty acid content examined. The composition of the monounsaturated fractions from these immature seeds was not different from that of the other samples which were examined in detail. The major C’,(, , fatty acids identified in evening primrose were n-7 and n-9, which contrasts to borage, which contains C‘,(, I (n-7) and C’,,,, (n-5) fatty acids (Wretetrsjo et ~1.. 1990). Gibson et rd. ( 1992) reported the presence of C,, , and C,, , fatty acids in commercial evening primrose oil samples which they attributed to adulteration of the oils, since they could not detect these acids in any of the seed samples they analysed; however, C,, , has been reported in evening primrose oil in other studies (Muderhu-a (V rrl.. 1987; Singer. 1990). The chromatograms of the monounsaturated fractions from the silicic acid silver nitrate columns of the individual samples examined did not consistently contain peaks with retention times corresponding to C! , and C,, i fatty acids. In fact, only one of the samples examined contained very small peaks corresponding to these two acids. The concentration and inconsistency of these peaks did not warrant further investigation. Linoleic [C,,:, (n-6)] and gamma-linolenic [C,, : (n-6)] acids were the major polyunsaturated fatty acids found in evening primrose oil samples (Fig. 1). Smaller amounts of C,, 1 (n-3). C’,? _i (n-3), and C,, 2 (n-6) were also found. In addition. the presence of a C,,:? and another C,,.: fatty acid were also detected by GCMS although the position of the double bonds was not determined. Flowering is a gradual process for evening primrose, with pod formation often occurring in Ontario from July until the plant is killed by frost. In addition, Lotti et al. (1984) found that temperature
185
Fatty acid and oil content qf’evrtning prirnrosr
19 23
-
0
ICI,, 1
1
2
10
3 *4
56
20
40
30
TIME (MINI Fig. 1. Chromatogram of the separation of fatty acids in evening primrose oil as methyl esters on a Quadrex CPS-I fused-silica capillary column. Operating conditions as in Materials and methods section. Peak identification: see Table 3.
during seed formation had an influence on the fatty acid content of the oil. In order to evaluate the extent that this observation had on the problems encountered in sampling single plants for oil and gamma-linolenic acid content, individual plants were subdivided into eight parts (Table 4). Analysis of the seed samples from the plant parts showed a range of approximately two per cent in both oil and gamma-linolenic acid content. The results indicate that the gamma-linolenic acid content of the seeds of the inner part of the stems which are formed during the warmer summer months have lower gamma-linolenic acid content than seeds on the outer part of the stem which were formed during the cooler fall temperatures. This agrees with the temperature effect found by Lotti et al. (1984). It contrasts with the observation of Yaniv et al. (1989) who observed that gamma-linolenic acid was higher in the pods maturing earlier with Oenothera lamarckiana. The variation in oil content was less consistent than gamma-linolenic acid but it was common for the seeds from the more mature inner parts of the
stem to have a higher oil content. The results clearly indicate that care must be taken in interpreting data obtained from individual plant selections unless the whole plant is sampled. Table 4. Evaluation of oil and gamma-linolenic acid content of different parts of mature evening primrose plants Plant position
1989
Oil
-_
-_
1990
C IX ? (n-6)
Oil
1991
C IS 3 (n-6)
c Oil
18.3
(n-6)
(%I) (‘56)”
(‘X,) (“A,)
(‘XI)
(‘X,)
25.5
13.7
22-5
1I .;
23-l
!3.8
Main stem ~~ inner
24.5
2.0
23.4
10
‘l-7
!2.4
Top
25.2
3.6
23.9
3.0
24.0
i3.0
27.0
2.8
23.5
I.6
23.8
12.9 12-9
Main stem -
tip
..~ tip
Top - inner Middle
25.3
3.4
22.1
34
23.9
Middle
~~inner
25.7
21
22.9
1.1
23-7
12.7
Bottom
-~ tip
25.6
3.4
22-6
Bottom
~-- inner
23-7 23-9
13.5 12.8
0.71
0.35
SI?
~ tip
25.9
11.9
33.6
2.0 IO.6
0.28
O-12
0.92
0 32
“Weight per cent of total fatty acids. “Standard error.
ACKNOWLEDGEMENT The authors gratefully acknowledge the financial assistance of the Ontario Primrose Producers’ Cooperative Ltd. REFERENCES Ackman, R. C. (1986). WCOT (capillary) gas-liquid chromatography. In Analysis of’Oils und Fats, ed. R. J. HamilIan & J. B. Rossell. Elsevier Applied Science Publishers, London, pp. 137-206. Baker, J. D. (1987). Parameters influencing the behaviour of evening primrose (Oenothera biennis). MS thesis, University of Guelph, Guelph, Ontario. Bottazzi, F., Izzo, R. & Lotti, G. (1985). Influenza de1 riscaldamento sulla composizione dell’olio di Ocnotheru hiennis L. Agrochimicu, 29, 331-40. Carter, J. P. (1988). Gamma-linolenic acid as a nutrient. Food Technology, 44, 72-82. Court, W. A., Roy, R. C. & Hendel, J. G. (1984). Effect of harvest date on agronomic and chemical characteristics of Ontario peanuts. Cun. J. Plant Sci., 64, 521-S. Craig, B. M. & Bhatty, M. K. (1964). A naturally occurring all-cis 6,9,12,15-octadecatetraenoic acid in plant oils. JAOCS, 41, 209-l 1. De Vries, B. (1963). Quantitative separations of higher fatty acid methyl esters by adsorption chromatography on silica impregnated with silver nitrate. JOACS, 40, 18&6. Gibson, R. A., Lines, D. R. & Neumann, M. A. ( 1992). Gamma linolenic acid (GLA) content of encapsulated evening primrose oil products. Lipids, 27, 824. Hall, I. V., Steiner, E., Threadgill, P. & Jones, R. W. (1988). The biology of Canadian weeds. 84 Oenothera hiennis L. Gun. J. Plant Sci., 68, 163-73.
Hudson. B. .I. F. (1984). Evening primrose (Orr~othc~tr hpp I oil and seed. JAOCS, 61. 540-43. Lotti. G.. Izzo, R. & Marchini, F. (1980). La iomposlzmnc acidica dell’olio di semi di Oenotheru hienni~ I.. durante la maturazione. Agrochemicu. 24, 274-85. Lotti, G.. Izzo. R., Bottazzi. F. & Bellani. A. 1 ( 1984). lnfluenza dell’epoca di semina sul &lo di sviluppo della pianta e sulle caratteristiche. dell’olio di Ocnc~ih~~vrrhicw7i.s L. Agrucher?~ica, 28. 85~95. McLafI’erty. F. W. & Staufer, D. B. (1989). I‘/zc W’ile,vNB.S Registry ~j’Mus.s Spectra Tutu. John Wiley. New York. Muderhwa. J. M., Dhuique-Mayer, C.. Pina. M.. Galzy. P.. Grignac, P. & Graille. J. (1987). Repartition interneiexterne des acides gras des triglycCrides de quelques huiles gamma linoleniques. Olbugineur, 42. 207~~11. Singer, P.. Moritz. V.. Wirth. M.. Berger. 1. & Forstcr, D. (1990). Blood pressure and serum lipids from SHR after diets supplemented with evening primrose, sunflower seed or fish oil. Pro,stu&ndins LcLkotrirnrs urrtl I%wntiul Fut t 1‘ Ads. 40, 17 20. Willner. D. (1965). Separation of fatty acid ester\ on acidtreated florisil impregnated with silver nitrate. (‘ilcn~. Lzud Id. 1839 1940. Wolf, R. B.. Kleiman, R. bi England, R. I:. (1983). New sources of y-linolenic acid. J. ,4nz. Oil C’hcm Sk.. 60, 1858MO. Wretensj8. I., Svensson. L. & Christie, W. W. (1990). Gas chromatographic-mass spectrometric identification of fatty acids in borage oil using picolinyl ester derivatives. J. Chmmutogr., 521, 89-97. Yaniv, Z., Ranen, C., Levy, A. & PaLevitch. D. (1989). Effect of temperature on the fatty acid composition and yield of evening primrose seeds (Oenotheru lamurtkiunu). J. E.xp. Bot.. 40, 609%13.
(Received 27 August 1992: accepted 1992)
I
December
26 (1993) 181-186
Determination of the fatty acids and oil content of evening primrose (Oenothera biennis IL.) William A. Court, John G. Hendel Chemistry
Luhoratory.
Agriculture
Canada Research Station,
& Robert Pots
PO Bos 186. Delhi, Ontario, Cunada N4B 2 WY. *
A rapid method for the analyses of oil content and individual fatty acids of evening primrose (Oenothera hiennis L.) is described. Oil was extracted from the seed with a Polytron probe generator using petroleum ether. A portion of the extract was concentrated to determine oil content. Results obtained with the Polytron extraction procedure were comparable to those obtained with a Soxhlet extraction procedure using the same solvent. Saponification of the oil with methanolic sodium hydroxide, methylation with methanolic sulfuric acid, and extraction of the esters with hexane was accomplished in a single reaction vial. Quantitation of the extracted esters was carried out by capillary gas chromatography. A number of fatty acids were identified by gas chromatographic-mass spectrometric studies of the extracted esters and methyl esters separated by their degree of unsaturation with silver nitrate-silicic acid chromatography. Saturated fatty acids from C,, to Cz4 were present in the oil with the even numbered acids present in the larger amounts. Eight monounsaturated fatty acids from C14,, to C,,: , were identified with C,, , (n-9) as the major constituent of this fraction. Seven polyunsaturated fatty acids were identified with C,,:, (n-6) and C,,:, (n-6) as the major component. Analysis of seeds from different parts of evening primrose plants showed that differences in gamma-linolenic [C,, : (n-6)] acid and oil content exist due to location of the seed pods. Keywords:
Oenothera
gas chromatography,
biennis, oil, fatty acids, gamma-linolenic gas chromatography-mass spectrometry.
INTRODUCTION
large amounts of gamma-linolenic acid (Baker, 1987). Analyses of individual plant selections for oil content and individual fatty acids requires a method that minimises seed utilisation; however, an unexpected problem encountered by local producers was unreliable and unreproducible analyses of individual plant selections and commercial crops for gamma-linolenic acid and oil content which has been previously reported (Hudson, 1984). In addition, differences have been reported in the minor fatty acids present in evening primrose seed (Lotti et al., 1984; Bottazzi et al., 1985; Muderhwa et al., 1987; Singer et al., 1990, Gibson et al., 1992). Since it is possible that these differences are due to genetic differences, an examination of the minor fatty acid components of these local plant types would be useful. The object of the study was to develop a fast and accurate method for the determination of oil and individual fatty acid content of evening primrose seed which minimises seed utilisation, and
Evening primrose (Oenothera biennia L.) is a common weed that is native to North America. The species is drought tolerant and grows well on sandy soils (Hall et al., 1988). Evening primrose is of interest because the seeds contain an oil that is rich in gamma-linolenic acid. Although this essential fatty acid has been found in other oilseeds, evening primrose is the most common commercial source (Wolf et al., 1983). Evening primrose is cultivated in a number of countries and because of possible nutritional and pharmaceutical applications, the demand for the oil continues to grow (Carter, 1988). Evening primrose is produced in Ontario, Canada using biotypes selected from local wild populations because these plant selections contain *Contribution
acid, capillary
number 223.
Food Research International 0963-9969/93/$06.00 0 1993 Canadian Institute of Food Science and Technology 181
which is capable of handling the large sample numbers generated by production and plant breeding research programs. In addition the study was conducted to identify the fatty acids present in the individual plant selections found in the region which are used for commercial production.
imately 5 min with dry nitrogen. ‘The weight IIIcrease in the vial was used to calculate the pcrcentage of oil extracted. Corrections wcrc made for the amount of solvent. the internal standard. and the volume change due to the oil extracted. Fatty acid analysis
MATERIALS AND METHODS Reagents and materials
Standard fatty acids and methyl esters were obtained from Sigma Chemical Co., St Louis, MO, Supelco Canada Ltd, Oakville, ON, and Mandel Scientific Co., Guelph, ON. Solvents were obtained from Caledon Laboratories, Georgetown, ON. Except for methanol, which was dried with magnesium, solvents were glass distilled before use. Except for silver nitrate which was obtained from Fisher Scientific Toronto, ON, all other reagents were obtained from J.T. Baker Chem Co., Toronto, ON. Silicic acid (BIO-SIL A) was obtained from Bio-Rad Laboratories, Mississauga, ON. Evening primrose, borage and canola seed were grown at the Agriculture Canada Research Station, Delhi, ON. All seed samples were hand cleaned prior to extraction. Moisture determination
The moisture content of evening primrose seed was determined by heating a sample in a convection oven for 3 h at 130°C. Oil extraction
A 250 mg sample of evening primrose seed was transferred to a tared 25 ml scintillation vial. Ten millilitres of petroleum ether (boiling point 40-60°C) containing the internal standard (tridecanoic acid) were added. The mixture was homogenised for 3 min with a Polytron PTl OST (Brinkmann Inc., Rexdale, ON) probe generator. Periodically during the extraction and after completion the sides of the vial were washed with small portions of petroleum ether. In addition, the probe generator was thoroughly rinsed with ether after homogenisation. After petroleum gently mixing, the vial was capped and the solid material allowed to settle. A 7 ml portion of the micella was then transferred to a tared 3 g sample vial. The solvent was allowed to evaporate in a fume hood with the last traces removed in approx-
Fatty acids were determined by saponifying the oil in the 3 dram sample vial with 2 ml of 0.5 N NaOH in dry methanol at 70°C for 40 min (Court et al.. 1984). After cooling. 1.5 ml of .5’::1H,SO, in dry methanol were added and the mixture reheated at 70°C for 30 min. After cooling, 2.5 ml of saturated NaCl and 4 ml of hexane were added. The vial was shaken for 2 min and the phases allowed to separate. A portion of the hexane layer was rcmoved and analyzed by gas chromatography. Silicic acid-silver nitrate chromatography
Fatty acid methyl ester mixtures were separated according to the degree of unsaturation with 10% silver nitrate on silicic acid according to De Vries (1963). The separation of 650 mg of esters was carried out with 65 g of absorbent. The elution of the methyl esters was with benzene/petroleum ether/ether mixtures (De Vries, 1963; Craig & Bhatty, 1964) or hexane/ether mixtures (Willner, 1965). Gas chromatography Quadrex C’PS- I
Quantitative analyses were performed on a Hewlett-Packard 5890 gas chromatograph equipped with dual FID detectors. A Quadrex CPS- 1 (Quadrex Corp., New Haven. CT) fused silica capillary column (25 m X 0.25 mm i.d., film thickness of 0.25 pm) was employed. One-microlitre injections were made with a HewlettPackard 7672A autosampler. The column oven was temperature programmed from 120 to 200°C at 3”Umin and held to 200°C for 4 min. A slightly longer final time was required if the CZli methyl ester was to be determined. The injector temperature was 183°C detector 200°C. and helium flow was 16.9 cm/s. The split (1 : 45) injection mode was utilised. Supelco
Supelcowllx
10
Analyses were performed in a Hewlett-Packard 5840 gas chromatograph equipped with dual FID detectors. A Supelco Supelcowax 10 (Supelco Canada
Fatty acid and oil content qf evening primrose
183
Ltd, Oakville, ON) fused silica capillary column (60 m X 0.25 mm i.d., film thickness 0.25 pm) was employed. The column oven was temperature programmed from 190 to 240°C at 2”C/min and held at 240°C for 35 min. The injector temperature was 225°C detector 225°C and helium flow was 16.7 cm/s. The split (1 : 50) injection mode was utilised.
Table 1. Comparison of procedures for the determination of oil content of evening primrose
Gas chromatography/mass spectrometry
i
GC/MS analyses were performed on a HewlettPackard 5971 MSD system equipped with a Quadrex CPS-1 (25 m X 0.18 mm i.d.) fused silica capillary column (film thickness 0.25 pm). Onemicrolitre injections were made with a HewlettPackard 7673A autosampler. The splitless injection mode was utilised. The column oven was temperature programmed as follows: 120°C for 1 min, 3”/min to 190°C and held at 190°C for 45 min. The injector temperature was 185°C and helium flow 16 cm/s. Sample components were tentatively identified by mass spectra matching with a Wiley Mass Spectra data base (McLafferty & Stauger, 1989). Tentative identifications were verified by comparison of retention times and mass spectra with that of authentic reference standards where possible, or by previously characterised mixtures such as borage (Wretensjo et ul., 1990) and canola (Ackman, 1986).
Sample N0
Soxhlet extraction
Polytron
extraction
Mean” (‘%,)
SDh
CV’ (‘XI)
Mean” (‘X,)
SD
CV (‘%I)
26.5’ 26.3 24.3
0.18 0.08 0.10
o-7 0.3 0.4
26.7 ‘6-7 74.5
0,41 0.41 0.32
I.5 I.5 l-3
1 3
~~. _ .~.
~~~~~
“Mean value of six determinations. ‘Standard deviation. ‘Coefficient of variation. “Mean value of seven determinations. Values reported are in percentage on a dry weight basis.
tive standard deviation from the mean for the Polytron extraction procedure was greater than that for the Soxhlet procedure but was still quite acceptable. For routine analyses 250 mg of seed were used; however. this was not a limiting factor and smaller seed samples can be readily accommodated. Fatty acid methyl esters were separated and quantitated by capillary gas chromatography. Reproducibility studies for individual fatty acids with two representative evening primrose samples are given in Table 2. Seven individual samples were analysed to obtain this data. Except for several of the very minor fatty acids, acceptable reproducibility was obtained.
Influence of seed pod position
Table 2. Reproducibility data for replicate analyses of evening primrose fatty acids by gas chromatography
Plots were grown at the Agriculture Canada Research Station, Delhi, ON. Mature plants were hand harvested and divided into eight parts as shown in Table 4. Ten plants were harvested and combined to yield each sample. Two replications were employed for each sample.
Acid
RESULTS AND DISCUSSION A number of methods have been used for the determination of the oil content of evening primrose (Lotti rt ul., 1980; Bottazzi et al., 1985; Yaniv et al., 1989). Among these methods the Soxhlet extraction of ground seed has been used extensively. In order to evaluate the effectiveness of the Polytron extraction procedure for the determination of the oil content of evening primrose, it was compared to the Soxhlet procedure using petroleum ether as the extraction solvent (Table 1). Agreement between the two procedures was quite good; however, the rela-
Sample
c I6 C,, c‘,, C,: C,; C,, CP Cl, C,x C,, CIK C?,, C?,,
I (n-9)
i (n-7) / (n-8) I (n-9) I (n-7) I (n-6) i (n-6) 1 (n-3)
I (n-9) Czri 2(n-6) CZ CZl
1”
Sample 2”
“/;,h
CV” (%I)
// b
0.04 0.07 0.03 6.44 0.04 0.07 0.06 0.04 1.81 9.76 0.84 71.20 8.72 0.18 0.31 0.23 0.03 0.12 0.04
7.1 4.8 7 I 1.4 7.2 9.1 9.1 6.2 06 0.8 1.5 0.1 0.3 39 9-5
0.03 0.07 0.03 5.93 0.04 O-04 0.07 O-02 2.08 4.99 0.56 74.40
104 3.9 IO.0 46
‘lMean value of seven determinations. ‘Weight per cent of total fatty acids. ‘Coefficient of variation.
IO.80 0.17 o-31 0.23 0.05 0.13 O-06
cv (0%) 7.5 12-3 74 0.4 8.3 17.8 8.8 3-6 I.6 I.1 2-3 0.2 0.1 7.6 4.5 5.2 5.0 11-3 7.5
Table 3. Fatty acids of evening primrose (Oenotheva hiennis L.) wild biotypes Peak No.
Fatty acid
Saturated
I
c‘12 c’,J C,, C,, Clh C,T C,, C,, Czcr
Identification
SP-IO”
0.403 0,495 O-556 0,664 0,776 0.888
0.419 0.495 0.550 0,639 0.751 0,864 I~000 1.142 I.313 I.515 I.766 2.076 2.468
GC-MS’, X-MS, CC-MS, GC-MS. GC-MS, GC-MS, GC-MS. GC-MS, GC-MS. GC-MS, GC-MS. GC-MS, GC-MS,
RT” RT RT RT RT RT RT RT RT RT RT RT RT
0.567 0,658 0.773 0.780 0,899 I.035 I.358
GC-MS GC-MS GC-MS. GC-MS. GC-MS, GC-MS, GC-MS. CC-MS.
RT RT RT RT RT RT
0.972 I .086 I.115 I.158 I-207 I-264 I.455
GC-MS GC-MS GC-MS. GC-MS, GC-MS, GC-MS. GC-MS.
RT RT RT RT RT
I.109 I.215 I.357 I .442 I -629 I.774
C2l
fatty acids
C 14 I C I? I ;I6 , (n-9) Ih. I Crj I Cl*. I C,,, I c 2. ,
Polyunsaturated I2 I6 17 I8 20 21 25
time
C‘PS-I”
1.000
Czz CY c 24
Monosaturated 4 6 8 9 II 14 I5 24
retention
fatty acids
;7 5 I IO 13 19 23 26 27 28 29
Relative
(n-7) (n-8) (n-9) (n-7) (n-9)
0.564 0.670 0.793 0.802 0.910 1.021 I.029 I.234
I.042
fatty acids
c,,,, C,, 2 CIx.2 (n-6) C,,, (n-6) C,# 3 (n-3) C18.4 (n-3) C 2. 2 (n-6)
0.966 I.052
I.080 I.101 I.131 1.159 I.283
“Quadrex CPS-I capillary column (25 m X 0.35 mm i.d.). “Supelco Supelcowax IO capillary column (60 m X 0.25 mm i.d.). ‘Gas chromatography-mass spectroscopy. “Retention time.
In order to facilitate identification of the minor fatty acids present in evening primrose oil, the methyl esters were separated according to their degree of unsaturation by column chromatography on a silver nitrate-silicic acid absorbent (De Vries, 1963). Saturated fatty acids from C,, to Cl4 were present in the column fractions with the even numbered acids present in larger amounts than the odd numbered fatty acids (Table 3). Except for the C,,, CIy, C2,, and Cl3 acids, all the other saturated fatty acids have been reported previously in evening primrose (Lotti et al., 1980, 1984). A large number of monounsaturated fatty acids have been reported in evening primrose oil with various studies reporting different components
(Lotti et d., 1980; Bottazzi et LII., 1985: Gibson c’l ui., 1992). Examination of the oil samples and unriched column fractions by gas chromatography indicated the presence of a number of monounsaturated fatty acids with substantial amounts of eight monounsaturated fatty acids (Table 3). The major component of this fraction was C !, (11-9) fatty acid with C,, , and C,, , as very minor components which were only evident in the enriched column fractions. Bottazzi et al. t 1985) reported the presence of shorter monoene fatty acids under certain conditions: however, if present they were in very small amounts in the samples examined in this study. Lotti et rrl. (1980) reported that the fatty acid content of evening primrose oil was dependent on the ripeness of the seeds. In order to examine this possibility, seeds were collected IO weeks prior to harvest and their fatty acid content examined. The composition of the monounsaturated fractions from these immature seeds was not different from that of the other samples which were examined in detail. The major C’,(, , fatty acids identified in evening primrose were n-7 and n-9, which contrasts to borage, which contains C‘,(, I (n-7) and C’,,,, (n-5) fatty acids (Wretetrsjo et ~1.. 1990). Gibson et rd. ( 1992) reported the presence of C,, , and C,, , fatty acids in commercial evening primrose oil samples which they attributed to adulteration of the oils, since they could not detect these acids in any of the seed samples they analysed; however, C,, , has been reported in evening primrose oil in other studies (Muderhu-a (V rrl.. 1987; Singer. 1990). The chromatograms of the monounsaturated fractions from the silicic acid silver nitrate columns of the individual samples examined did not consistently contain peaks with retention times corresponding to C! , and C,, i fatty acids. In fact, only one of the samples examined contained very small peaks corresponding to these two acids. The concentration and inconsistency of these peaks did not warrant further investigation. Linoleic [C,,:, (n-6)] and gamma-linolenic [C,, : (n-6)] acids were the major polyunsaturated fatty acids found in evening primrose oil samples (Fig. 1). Smaller amounts of C,, 1 (n-3). C’,? _i (n-3), and C,, 2 (n-6) were also found. In addition. the presence of a C,,:? and another C,,.: fatty acid were also detected by GCMS although the position of the double bonds was not determined. Flowering is a gradual process for evening primrose, with pod formation often occurring in Ontario from July until the plant is killed by frost. In addition, Lotti et al. (1984) found that temperature
185
Fatty acid and oil content qf’evrtning prirnrosr
19 23
-
0
ICI,, 1
1
2
10
3 *4
56
20
40
30
TIME (MINI Fig. 1. Chromatogram of the separation of fatty acids in evening primrose oil as methyl esters on a Quadrex CPS-I fused-silica capillary column. Operating conditions as in Materials and methods section. Peak identification: see Table 3.
during seed formation had an influence on the fatty acid content of the oil. In order to evaluate the extent that this observation had on the problems encountered in sampling single plants for oil and gamma-linolenic acid content, individual plants were subdivided into eight parts (Table 4). Analysis of the seed samples from the plant parts showed a range of approximately two per cent in both oil and gamma-linolenic acid content. The results indicate that the gamma-linolenic acid content of the seeds of the inner part of the stems which are formed during the warmer summer months have lower gamma-linolenic acid content than seeds on the outer part of the stem which were formed during the cooler fall temperatures. This agrees with the temperature effect found by Lotti et al. (1984). It contrasts with the observation of Yaniv et al. (1989) who observed that gamma-linolenic acid was higher in the pods maturing earlier with Oenothera lamarckiana. The variation in oil content was less consistent than gamma-linolenic acid but it was common for the seeds from the more mature inner parts of the
stem to have a higher oil content. The results clearly indicate that care must be taken in interpreting data obtained from individual plant selections unless the whole plant is sampled. Table 4. Evaluation of oil and gamma-linolenic acid content of different parts of mature evening primrose plants Plant position
1989
Oil
-_
-_
1990
C IX ? (n-6)
Oil
1991
C IS 3 (n-6)
c Oil
18.3
(n-6)
(%I) (‘56)”
(‘X,) (“A,)
(‘XI)
(‘X,)
25.5
13.7
22-5
1I .;
23-l
!3.8
Main stem ~~ inner
24.5
2.0
23.4
10
‘l-7
!2.4
Top
25.2
3.6
23.9
3.0
24.0
i3.0
27.0
2.8
23.5
I.6
23.8
12.9 12-9
Main stem -
tip
..~ tip
Top - inner Middle
25.3
3.4
22.1
34
23.9
Middle
~~inner
25.7
21
22.9
1.1
23-7
12.7
Bottom
-~ tip
25.6
3.4
22-6
Bottom
~-- inner
23-7 23-9
13.5 12.8
0.71
0.35
SI?
~ tip
25.9
11.9
33.6
2.0 IO.6
0.28
O-12
0.92
0 32
“Weight per cent of total fatty acids. “Standard error.
ACKNOWLEDGEMENT The authors gratefully acknowledge the financial assistance of the Ontario Primrose Producers’ Cooperative Ltd. REFERENCES Ackman, R. C. (1986). WCOT (capillary) gas-liquid chromatography. In Analysis of’Oils und Fats, ed. R. J. HamilIan & J. B. Rossell. Elsevier Applied Science Publishers, London, pp. 137-206. Baker, J. D. (1987). Parameters influencing the behaviour of evening primrose (Oenothera biennis). MS thesis, University of Guelph, Guelph, Ontario. Bottazzi, F., Izzo, R. & Lotti, G. (1985). Influenza de1 riscaldamento sulla composizione dell’olio di Ocnotheru hiennis L. Agrochimicu, 29, 331-40. Carter, J. P. (1988). Gamma-linolenic acid as a nutrient. Food Technology, 44, 72-82. Court, W. A., Roy, R. C. & Hendel, J. G. (1984). Effect of harvest date on agronomic and chemical characteristics of Ontario peanuts. Cun. J. Plant Sci., 64, 521-S. Craig, B. M. & Bhatty, M. K. (1964). A naturally occurring all-cis 6,9,12,15-octadecatetraenoic acid in plant oils. JAOCS, 41, 209-l 1. De Vries, B. (1963). Quantitative separations of higher fatty acid methyl esters by adsorption chromatography on silica impregnated with silver nitrate. JOACS, 40, 18&6. Gibson, R. A., Lines, D. R. & Neumann, M. A. ( 1992). Gamma linolenic acid (GLA) content of encapsulated evening primrose oil products. Lipids, 27, 824. Hall, I. V., Steiner, E., Threadgill, P. & Jones, R. W. (1988). The biology of Canadian weeds. 84 Oenothera hiennis L. Gun. J. Plant Sci., 68, 163-73.
Hudson. B. .I. F. (1984). Evening primrose (Orr~othc~tr hpp I oil and seed. JAOCS, 61. 540-43. Lotti. G.. Izzo, R. & Marchini, F. (1980). La iomposlzmnc acidica dell’olio di semi di Oenotheru hienni~ I.. durante la maturazione. Agrochemicu. 24, 274-85. Lotti, G.. Izzo. R., Bottazzi. F. & Bellani. A. 1 ( 1984). lnfluenza dell’epoca di semina sul &lo di sviluppo della pianta e sulle caratteristiche. dell’olio di Ocnc~ih~~vrrhicw7i.s L. Agrucher?~ica, 28. 85~95. McLafI’erty. F. W. & Staufer, D. B. (1989). I‘/zc W’ile,vNB.S Registry ~j’Mus.s Spectra Tutu. John Wiley. New York. Muderhwa. J. M., Dhuique-Mayer, C.. Pina. M.. Galzy. P.. Grignac, P. & Graille. J. (1987). Repartition interneiexterne des acides gras des triglycCrides de quelques huiles gamma linoleniques. Olbugineur, 42. 207~~11. Singer, P.. Moritz. V.. Wirth. M.. Berger. 1. & Forstcr, D. (1990). Blood pressure and serum lipids from SHR after diets supplemented with evening primrose, sunflower seed or fish oil. Pro,stu&ndins LcLkotrirnrs urrtl I%wntiul Fut t 1‘ Ads. 40, 17 20. Willner. D. (1965). Separation of fatty acid ester\ on acidtreated florisil impregnated with silver nitrate. (‘ilcn~. Lzud Id. 1839 1940. Wolf, R. B.. Kleiman, R. bi England, R. I:. (1983). New sources of y-linolenic acid. J. ,4nz. Oil C’hcm Sk.. 60, 1858MO. Wretensj8. I., Svensson. L. & Christie, W. W. (1990). Gas chromatographic-mass spectrometric identification of fatty acids in borage oil using picolinyl ester derivatives. J. Chmmutogr., 521, 89-97. Yaniv, Z., Ranen, C., Levy, A. & PaLevitch. D. (1989). Effect of temperature on the fatty acid composition and yield of evening primrose seeds (Oenotheru lamurtkiunu). J. E.xp. Bot.. 40, 609%13.
(Received 27 August 1992: accepted 1992)
I
December