ANALYSIS OF GAMMA-LINOLENIC AND OTHER FATTY

ACIDS IN THE EDIBLE PART OF BORAGE (Borago officinalis L.).

M. del Ríoa, A. de Harob

a C.I.D.A. Apdo. 4240. 14.080 CORDOBA

b Inst. Agricultura Sostenible C.S.I.C. Apdo. 4084. 14080 CORDOBA.

ABSTRACT

Borage (Borago officinalis L.) is currently used as a vegetable in the North of Spain.

The edible parts of the plant are the basal leaf petioles, leaves and stem. The objective

of this study has been to determine the oil, gamma-linolenic acid and other fatty acids

content in the tissues of borage. The leaves were the most important source of lipid

with 9 % dry weight, while basal leaf petioles and stem were 1.8 % and 0.92 %

respectively. Alfa-linolenic acid and GLA are formed by desaturation of linoleic acid

by delta-6 and delta-15-desaturases, respectively, but the activity of the delta-15-

desaturase predominates. Alfa-linolenic was the major constituent with about 37% in

tissues photosyntetics and 24% in stem and basal leaf petioles. Basal leaves and main

stem leaves showed the highest content in GLA 15.8 % and 15.5% respectively. In

contrast to the seeds, all the tissues analyzed had stearidonic acid (18:4 n-3). The major

fatty acids were: palmitic, alfa-linolenic and stearidonic acids in leaves, and palmitic,

linoleic and alfa-linolenic acids in stem and basal leaf petioles. Inverse relation was

found between oleic acid and GLA and between linoleic vs. alfa-linolenic and

stearidonic acids in basal leaves. In main stem leaves and basal leaf petioles was also

found inverse relation between alfa-linolenic vs. oleic and linoleic acids.

.

INTRODUCTION

Gamma-linolenic acid (GLA, C:18, n-6,9,12, cis) is one essential fatty acid

precursor of indispensable compounds in the body such as prostaglandins (PGE1) and

leukotrienes (LT), often known as eicosanoids. Many factors have been shown to

interfere the bioconvertion of linoleic to GLA like ageing, stress, diabetes, high alcohol

intake, high cholesterol levels, viral infections and nutritional deficiencies. Since there

is some evidence that 6-desaturase activity is impaired in each of these situations, with

an exogenous supply of GLA these may be ameliorated [1]. Seeds of several plants

such as evening primrose (Oenothera biennis) and the common borage (Borago

officinalis) and the fruits of Ribes spp., have been found to contain substantial

proportions of GLA as a constituent of their lipids [2, 3, 4 ].

White flowered borage is currently used as a vegetable in the North of Spain, but no

attention has been paid till now to study as source of GLA. The parts of the plant that

are preferentially eaten are the basal leaf petioles (before the plant is completely

developed and before flowering) leaves and stem.

Lipids containing GLA are unusual constituents of tissues in higher plants, which are

generally rich in alfa-linolenic acid (all-cis-9,12,15-octadecatrienoic acid). Apparently a

plant that synthesizes substantial amounts of GLA acid in the seeds, should form this

acid in the leaves. However the leaves of the evening primrose plants contain alfa-

linolenic as major constituent of photosynthetic tissues but not GLA [5].

The objective of the present study was to determine the oil and fatty acids content in

the edible portion of the borage with emphasis on GLA acid.

MATERIAL

Seeds of 12 accessions of white flowered borage (B. officinalis L.) were provided by

The Center of Agricultural Research of Rioja . Borage plants were grown in a 16 h.

photoperiod at 15ºC and an 8 h. dark at 10ºC. Collections of the basal leaf petioles and

basal leaves were made before the plants were completely developed and before

flowering. The stem and main stem leaves were collected after flowering.

METHOD

Lipid Extraction and Analysis. Oil content was determined according to the

Association of Official Analytical Chemists. (AOAC, 1984, Method 7062) [6].

The fatty acid composition of oil was determined by GLC (Gas Liquid

Chromatography) of the corresponding methyl esters, using as quantifier

methylheptadecanoic acid as an internal standard . Digestion, transmethylation of lipids

from leaves, petioles and stem and extraction of fatty acid methyl esters in one step was

realized according to Garcés and Mancha [7]. The samples were heated at 80ºC with a

reagent containing methanol: heptane: toluene: 2-2-dimetoxypropane: H2SO4

(37:36:20:5:2). After 120 min. two phases were formed, the upper one containing the

fatty acid methyl esters ready for GLC analysis. The analysis were performed using a

Perkin Elmer Autosystem Gas Chromatograph equipped with flame ionization detector

and split injector. The chromatograph is equipped with a capillary column (25m x 0.25

mm, i.d. 0.25 mm film) with acidified polietilenglycol as the stationary phase. Oven

temperature was programmed from 190ºC to 210ºC at a rate of 2ºC/min. The

temperature of the detector and injector were 275ºC and 200ºC respectively. Nitrogen

was used as carrier gas.

RESULTS

The data given in Table I show the oil and fatty acids content in the portion edible of

borage in comparison with the content of seeds [8]. Leaves showed the highest content

of oil with a mean value of 9% dry weight. Basal leaf petioles and stem had a mean

value of 1.8% and 0.92% respectively. The major fatty acid in all the tissues. was alfa-

linolenic acid . Fatty acids composition was very similar in basal leaves and main stem

leaves of borage plant. The leaf lipids contained as the major constituents, about 15%

palmitic acid, 37% alfa-linolenic and 17% steridonic acids. Nevertheless, basal leaf

petioles and stem showed as the major fatty acids, about 20% palmitic acid, 19%

linoleic acid and 24% alfa-linoleic acid.

The GLA was present in all the tissues analyzed. The basal leaf petioles and stem

showed the highest content of GLA with a mean value of 15.8 and 15.5 respectively.

In contrast to the borage seeds, stearidonic acid was synthesized in substantial

amounts in all the tissues evaluated, with the highest levels, 17.04 and 18.72 in basal

and main stem leaves. Short-chain fatty acids such as lauric and miristic acids and

traces of C20-C24 fatty acids were found in the tissues evaluated.

Inverse relation was found between oleic acid and GLA and between linoleic vs. alfa-

linolenic and stearidonic acids in basal leaves. In main stem leaves and basal leaf

petioles was also found inverse relation between alfa-linolenic vs. oleic and linoleic

acids.

DISCUSSION

In contrast to the borage seeds, the lipids of basal leaf petioles, leaves and stem of

plant contained two n-3 acids alfa-linolenic and stearidonic acids. Alfa-linolenic acid

and GLA were detected in all the tissues of borage, which indicates that both delta-15

and delta-6 desaturases are active, but the effect of the delta-15 desaturases

predominates. Therefore alfa-linolenic is the major octadecatrienoic acid in the tissues

of borage. These results support that alfa-linolenic acid is channeled almost exclusively

to the phospholipids and glycolipids, which are the major constituents of the cellular

membranes. Whereas GLA is esterified mainly in the storage lipids, i.e triacylglicerols

of borage seeds, such as occur with evening primrose plant [9]. New studies are being

realized in our group of investigation for determining the proportions of these lipids in

the tissues of borage.

The inverse relation found between GLA and oleic acid in basal leaves suggests that

GLA is formed to desaturation of oleic acid., which corroborates the hypothetical

biosynthetic pathways to fatty acids in borage seeds that proposes Gunstone,[10] and

corroborates results obtained by Cherry et al. [11] and Whipkey et al. [12], in which the

sequence of alternating desaturations from oleic acid to GLA is significant. However,

others patterns of desaturations must be considered for explain the n-3 acid synthesis.

Considering the inverse relation found in basal leaves, between linoleic acid vs. alfa-

linoleic and stearidonic acids, as well as in main stem leaves and basal leaf petioles

between alfa linoleic acid vs. oleic acid and linoleic acid the results are compatible with

the following sequential desaturations:

oleic acid ® linoleic acid ® gamma-linolenic acid ¯ alfa-linolenic acid (18:3, n-3). ¯ stearidonic acid (18:4, n-4).

Additional works are being realized in order to explain the probable biosynthetic

pathways to fatty acids in borage.

ACKNOWLEDGMENTS

The authors wish to extend their appreciation to Dr. Vicente Dominguez (Centre of

Agricultural Research of Rioja) for providing the accessions of borage.

REFERENCES

1 D. F. Horrobin. Plant Lipid Res. Vol. 31, No. 2, (1992) 163.

2 B. J. F. Hudson. J. Am. Oil Chem. Soc. 61 (1984) 540.

3 H. Traitler, H. Winter, U. Richli and Y. Ingenbleek. Lipids. 19 (1984) 923.

4 R. B. Wolf, R. Kleiman and R. E. England. J. Am. Oil Chem. Soc. 60 (1983)

1858.

5 K. D. Mukherjee and I. Kiewitt. J. Agric. Food Chem., Vol. 35, (1987) 1009.

6 AOAC . Official Methods of Analysis, Williams, Arlington, 1984.

7 R. Garcés and M. Mancha. Analytical Biochemistry, Vol. 211 , (1993) 139.

8 M. del Río, J.M.Fernández and A. de Haro. Grasas y aceites. 44 (1993) 125.

8 G. , Lotti and M. F., Quartacci. Agrochimica. 34 . (1990) 243.

9 F. D. Gunstone. Plant Lipid Res. Vol. 31, No. 2, (1992) 145.

10 J. H. Cherry, L. Bishop, P. M., Hasegawa and H. R., Leffler. Ibid. 24 .(1985). 237

11 A., Whipkey, J. E. Simon and J. Janick. JAOCS. 65. (1988) 979.

TABLE I

Fatty Acid Composition and oil content of the edible portion of borage (data are given as peak area percentages).

oil content

a

12:0 b 14:0 16:0 16:1 18:0 18:1 18:2

main stem leaf 93.740.92-

8.24

0.180-0.79

14.3012.42-18.08

1.60.31-4.52

2.951.54-5.63

3.061.13-5.67

9.667.67-11.74

basal leaf 93.31

0-8.011

0-8.715.73

12.46-18.191.13

0-1.823.07

2.06-4.493.24

1.01-6.159.34

7.04-11.14

basal leaf petioles 1.8 ----------0.89

0-2.3420.9

19.09-21.911.63

0-3.843.59

0-7.153.78

0-8.4417.86

13.14-20.98

stem 0.91 ----------1.37

0-2.1321.11

17.39-26.581.41

0.5-2.041.87

0-4.282.23

0-5.2621.05

16-97-28.17

seed 34.3 ---------- ---------10.88

8.5-18.7 -----------3.86

2.4-9.218.43

9.9-30.238.12

26.7-44.5

a Oil content is given as percentages of dry weight.

b Fatty acids are designated by number of carbon atoms:number of double bonds. c The n-6 (or omega-6) and the n-3 (or omega-3) define the position of the first

double bond in the molecule starting from the carbon atom at the methyl of the

chain.dIncluding 20:0, 20:1, 22:0 and 22:1.eIncluding 20:1 and 22:1.