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An Analytical Study on Fatty Acids in Pollen Extract

T. Seppanen and I. Laakso

Division of Pharmacognosy, School of Pharmacy, University of Helsinki, SF-00170 Helsinki, Finland

J. Wojcicki and L. Samochowiec

Institute of Pharmacology and Toxicology, Medical Academy, Powstancow Wielkopolskich 72, PL-70-11 Szczecin, Poland

Fatty acids in the fat-soluble fraction of pollen extract (Cernitin™ GBX™) were analyzed. Fatty acids were determined on a Dani 3860 PTV GC. Identification was based on the retention times of known mixtures of free fatty acids and their methyl esters in GC/MS. The major part of the fatty acid fraction was in free form. Bound fatty acids were characterized by a high content of a-linolenic acid (70%). The mechanism of antiatherosclerotic action of this pollen extract may be, at least in part, due to polyunsaturated fatty acids.

Keywords: Pollen extract, Fatty acids.

Introduction

Reports on the serum lipid-lowering effect of orally administered pollen extracts to rats (Samochowiec and Wojcicki, 1981; Wojcicki and Samochowiec, 1984) have been confirmed in humans (Wojcicki et al.,1983).

Pollen extracts-Cernitin™ T60™ and Cernitin™ GBX™ (AB Cernelle, Vegeholm, Sweden) were taken from six plant species: rye grass, maize, timothy grass, pine alder flower, and orchard grass. Cernitin™ T60™ contains water-soluble substances (6.0-9.2% of a-amino acids) while those in Cernitin™ GBX™ are mainly fat-soluble (10-16% phytosterols).

The chemical composition of pollen has been investigated (Kvanta, 1968; Nielson et al., 1957; Lelson and Holmstrom, 1957). Numerous chemical substances have been identified and isolated: 21 amino acids, all known vitamins, enzymes, coenzymes, sterols, minerals and trace elements.

This study was to analyze the fatty acids in the fat-soluble fraction of pollen extract (Cernitin™ GBX™) with regard to its proven antiatherosclerotic activity (Wojcicki et al., 1986).

Materials and Methods

The fatty acid composition of the fat-soluble pollen extract (Cernitin™ GBX™) was analyzed by gas chromatography. Bound fatty acids were transesterified by modifying the method of Hiltunen et al., (1979) as follows:

A sample (100 mg) of the fat-soluble pollen extract (batch No 759) was dissolved in 1mL petroleum spirit (b.p. 40-600C), transmethylated with 0.5mL 0.5 N NaOMe at 400C for 5 min and neutralized with 1mL of 15% NaHSO4. Petroleum spirit was added and 1µL taken from the upper layer for gas chromatography. Fatty acids were determined on a Dani 3860 PTV GC as follows: column OV-351 Nordion fused silica (25 m, 0.32 mm ID) oven programmed from 1000C at 100/min to 2250C, programmed temperature vaporizer (PTV)-injector from 700 to 2500C, carrier gas (H2) 0.8 bar, detector (FID) 2500C, sampling mode split (40:1). Identification was based on the retention times of known mixtures of free fatty acids and their methyl esters. Analyses after transesterification of triolein confirmed that no free fatty acids were formed under the conditions used. Other constituents such as aliphatic hydrocarbons and alcohols were identified by GC/MS.

Results and Discussion

GLC analyses of the fat-soluble pollen extract revealed that the major part (more than 60%) of the fatty acid was in the free form (Table 1, Fig. 1). Bound fatty acids, which rather reflect the compositional profile of pollen, were characterized by a high content of a-linolenic acid (18: 3n-3, a-LLA) (70%) followed by small amounts of linoleic (18: 2n-6) and oleic acid (181n-9) only. Palmitic acid (16:0) was the most abundant saturated

Previous studies have revealed that the pollen extract has beneficial properties, lowering serum lipid levels, reducing atherosclerotic plaque intensity (Wojcicki et al., 1986) and decreasing platelet aggregation both in vitro (Kosmider et a1.,1983) and in vivo (Wojcicki et al., 1983). If fatty acids are involved in these effects, the role of a-linolenic acid as a precursor of eicosapentaenoic acid (20: 5n-3, EPA) is significant, since EPA is considered to be responsible for reduced platelet aggregation (Dyer-berg and Bang, 1979). EPA in vivo is incorporated into platelet phospholipids, to some extent replacing arachidonic acid and exerting an antithrombotic effect either by competing with remaining arachidonic acid for cylco-oxygenase and lipoxygenase or by being converted to less proagreggatory PGH3 and TXA3 (Moncada and Vane, 1984). Studies in humans suggest that a diet supplemented with polyunsaturated Figure 1. GC chromatogram of pollen extract fatty acids on OV-351 column. Peak numbering as in Table 1. *Aljphatic hydrocarbons and/or alcohols.

Fatty acids decreases whole blood viscosity, and reduces triglyceride and cholesterol levels in patients with cardiovascular disease (Saynor et al., 1984). Recent clinical observations are in favour of a linolenic acid supply, leading to higher levels of phospholipid eicosapaentoic and docosahexaenoic acids (Jacotot et al., 1986). The metabolic conversion from a-LLA into EPA, which is known to occur in humans (Budowski et al., 1984; Sanders and Younger, 1983), would at least in part explain the mechanism of antiatherosclerotic action of pollen extract (Wojcicki et al, 1986).

Figure 1.

Figure 2.

References

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Received 15 September 1988. Accepted 1 November 1988.