pinolenic acid as a new source of phyto-polyunsaturated fatty acid

Lipid Technology June 2013, Vol. 25, No. 6 135

Feature

Pinolenic acid as a new source of phyto-polyunsaturated fatty acid

Da Som No, and In-Hwan Kim

D.S. No is a graduate student and I-H. Kim is a professor at the Department of Food and Nutrition, Korea University, Seoul,136-703, Republic of Korea. E-mail: [email protected]

Summary

Pinolenic acid (PLA, all-cis-5,9,12-18:3) is an interesting plant-based polyunsaturated fatty acid of which little is known. The major sourceof PLA is pine nut oil. PLA has distinctive health benefits such as LDL/VLDL cholesterol lowering potential and displays an appetitesuppressing effect. Enrichment of PLA can be carried out by physical procedures or by enzymatic reactions with lipases such as Candidaantartica lipase B and Candida rugosa lipase.

Introduction

Pinolenic acid (PLA) is a plant-source polyunsaturated fatty acidformally designated as all-cis-5,9,12-18:3. For common unsatu-rated fatty acids containing more than 1 double bond in themolecule, each double bond is one methylene group apart fromothers. For example, a-linolenic acid has three double bonds atD9, 12 and 15 positions. On the other hand, for pinolenic acid,the first and second double bond from the carboxyl group aretwo methylene groups apart. As one of the small D5-unsaturatedpolymethylene-interrupted fatty acid (D5-UPIFA) class, it has dis-tinctive characteristics differing from the more familiar 18:3fatty acids (alpha and gamma linolenic).

The primary source of PLA is pine nut oil. Pine nuts are theedible seeds of pinaceae. In conifer seeds, the fatty acids contentvaries depending on the botanical family considered. Amongthem, Pinacae seed oils contain several D5-olefinic acids includ-

ing 5,9-18:2, 5,9,12-18:3, 5,9,12,15-18:4, 5,11-20:2 and 5,11,14-20:3 acids (Figure 1). On the other hand, 5,11,14,17-20:4 acid isfound exclusively in Cupressaceae and Taxodiaceae seeds. Thefamily of Pinaceae contains a total of 11 accepted genera. Pinus isthe largest and most heteromorphic genus in the family of Pina-ceae, almost exclusively distributed in the Northern Hemi-sphere. This genus is ecologically versatile, and its phytogeogra-phical distribution is wide. Depending on the species in genuspinus, fatty acid composition, especially the content of D5-olefi-nic acids varies (Table 1). For example, the species in the Sylves-tres subsection exhibits very high D5-UPIFA percentage rangingbetween 25 and 30%, distributed between taxoleic (ca. 2-5%),pinolenic (ca. 17–23%), and sciadonic (ca. 2–6%) acids. On theother hand, less then 1% of D5-UPIFA concentration is observedin the species in the Cembroides subsection. One of the most com-mercially available sources of pine nut, P. koraiensis has ca. 17–18% of D5-UPIFA, including 13–15% of PLA. In general, pine spe-

i 2013 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim www.lipid-technology.com

DOI 10.1002/lite.201300278

Figure 1. D5-olefinic acids that occur in the seed lipids from selected gymnosperms, and their trivial names.

136 June 2013, Vol. 25, No. 6 Lipid Technology

cies from warm regions, such as P. merkusii, P. kremfii, P. dalatenis(from southeast Asia), P. edulis (from southwest of the UnitedStates and Mexico), P. pinea (a circum-Mediterranean pine) havesignificantly less D5-olefinic acids than most other pine speciesthat grow in high-altitude or in colder regions. Perhaps this isindicative of some relationship between the D5-unsaturationand resistance to cold [1].

The fatty acid distribution in triacylglycerols (TAG) has dis-tinctive features depending on species in the conifers. D5-UPI-FAs, especially PLA, are almost exclusively esterified to the sn-3position of TAG from several seeds from conifer species (Table 2).For example, for Pinus koraiensis, which contains ca. 14% of PLA,more than 97% of the PLA is esterified to the sn-3 position ofTAG. Also, more than 96% of total D5-UPIFAs are esterified to thesn-3 position. For Pinus pinaster, which contains ca. 6% of PLA,more than 89% of PLA is esterified to the sn-3 position of TAG.Additionally, more than 86% of D5-UPIFAs are esterified to thesn-3 position. This leads us to conclude that there is only onemolecule of D5-UPIFA in individual TAG and that this is at the asn-3 position, thus, total D5-UPIFA never exceeded 34% of totalfatty acids in the seeds from about 70 different conifer species[2].

Beneficial health effect

PLA is an isomer of c-linolenic acid (GLA), which is x-6 essentialfatty acid. Even though PLA is not classified as an essential fattyacid, it plays similar roles to essential fatty acids. It forms biolo-gically active metabolites in the presence of cyclooxygenase orlipoxygenase and these metabolites can partially relieve some ofthe symptoms of essential fatty acid deficiency. Moreover, PLAhas distinctive health benefits possibly coming from its doublebond at the D5 position.

First of all, PLA has a lipid-lowering effect, consequently lower-ing the risks of dyslipidemia. The mechanisms of this have notyet been revealed completely but several hypotheses have beensuggested. One of them is that PLA exerts part of its lipid-lower-ing effects by altering the expression of various apo genes. Apo-proteins have a crucial role in lipid metabolism. ApoA-I andapoA-II are key proteins of HDL metabolism. ApoE is necessaryfor lipoprotein remnant clearance. ApoC-III is a major compo-nent of triacylglycerol -rich lipoparticles and interferes withVLDL lipolysis and uptake by a cellular receptor. In the case ofPLA, ingestion reduces serum levels of LDL by enhancing hepaticLDL uptake. Another mechanism suggested is that PLA contri-


Table 1. Fatty acid composition (mol% of total fatty acids) of seed lipids from Pines of the subsection Strobi, Cambrae, Cembroides, Sylvestres

Fatty acid Strobi Cembrae Cembroides Sylvestres

P.strobus

P.armandii

P.cambra

P.sibirica

P.koraiensis

P.edulis

P.monophylla

P.sylvestris

P.nigra

P.merkusii

P.pinaster

16:0 3.9 4.5 4.3 4.3 4.7 7.1 6.9 3.4 4.2 5.3 4.618:0 1.9 1.9 2.7 2.4 2.1 2.3 3.2 1.8 2.0 4.6 2.718:1(D9) 13.8 23.9 23.2 25.1 27.3 46.9 46.1 14.4 17.6 16.5 18.918:1(D11) 0.4 0.5 3.0 0.3 1.1 0.6 0.4 0.8 0.7 0.5 0.418:2(D5,9) 1.7 3.4 1.7 2.0 2.0 0.1 – 2.7 3.6 1.7 0.918:2(D9,12) 46.7 46.0 44.7 43.7 44.7 40.7 41.2 44.8 45.0 54.9 52.218:3(D5,9,12) 25.3 16.0 19.2 18.5 14.5 0.4 0.1 21.7 18.9 10.3 7.918:3(D9,12,15) 0.2 0.2 0.3 0.2 0.1 0.2 0.2 0.4 0.6 1.3 1.420:0 0.3 0.4 0.3 0.3 0.4 0.5 0.4 0.2 0.3 0.4 0.320:1(D11) 1.2 0.9 1.2 1.2 1.2 0.5 0.5 1.1 1.0 0.4 1.220:2(D5,11)) 0.2 0.1 0.1 0.1 0.1 – 0.1 0.5 0.3 0.3 0.920:2(D11,14) 1.2 0.6 0.6 0.6 0.5 0.2 0.2 1.0 0.9 0.8 0.920:3(D5,11,14) 1.9 1.3 1.1 1.0 0.9 0.3 0.3 5.5 3.4 2.6 7.020:3(D7,11,14) 0.5 0.1 0.1 0.1 – – – 0.7 0.4 – 0.2SD5 29.1 20.8 22.1 21.6 17.5 0.8 0.5 30.4 26.2 14.9 16.7

Table 2. Fatty acid composition of trigacylglycerols (TAG) from the seeds of conifer species and fatty acid distribution in the sn-1, sn-2 and sn-3 posi-tions of TAG (results expressed as mol%)

Fatty acid Taxus baccata Larix decidua Juniperus communis Pinus koraiensis Pinus pinaster

sn-1 sn-2 sn-3 TAG sn-1 sn-2 sn-3 TAG sn-1 sn-2 sn-3 TAG sn-1 sn-2 sn-3 TAG sn-1 sn-2 sn-3 TAG

16:0 10.8 0.7 3.8 3.5 9.7 1.5 9.9 3.3 17.3 0.5 2.9 4.6 17.9 0.7 8.3 5.5 15 1.1 10.5 5.818:0 10.0 0.7 3.0 3.3 4.0 0.8 2.0 1.4 8.4 0.2 1.9 2.2 5.3 0.5 3.7 2.2 5.7 0.5 6.5 2.718:1(D9) 61.8 77.4 33.7 57.1 25.8 24.5 6.5 18.2 9.6 15.6 4.6 8.2 24.1 29.8 16.1 26.8 27.3 28.2 25.7 27.218:1(D11) 0.9 0 0 0.3 1.0 0.4 0 0.8 0.9 0 0.2 0.2 0.9 0.1 0.4 0.4 1.1 0 0.9 0.518:2(D5,9) 0.7 1.1 31.2 11.0 0.2 1.0 5.2 2.4 0 0 0 0 –0.3 0.3 5.3 2.0 1.0 0 2.3 0.818:2(D9,12) 13.0 18.5 21.8 20.0 51.3 64.9 6.7 43.1 29.4 56.8 19.7 34.1 50.1 66.4 12.7 45.0 45.6 65.7 19.3 46.718:3(D5,9,12) 0.3 0 0.9 0.4 5.6 5.5 68.2 28.8 0 0 1.0 0.3 0.7 1.0 50.2 15.5 1.1 0.9 16.8 7.318:3(D9,12,15) 1.1 1.0 1.3 1.5 0.7 0.4 0.2 0.6 23.4 16.2 20.0 21.5 0.5 0.2 0.4 0.1 1.5 0.7 1.2 1.420:0 0 0.1 0 0 0.3 0 0.5 0.1 1.0 0 0.4 0.4 0.2 0 0.6 0.3 0.6 0 0.6 0.220:1(D11) 1.1 0.4 0.6 1.1 0.4 0.3 0 0.3 1.7 0 0.2 0.8 0.8 0 1.1 0.9 0.6 0.3 1.0 0.820:2(D5,11)) 0 0 0.3 0.1 0 0 0.2 0 0 0.3 0.5 0.3 0 0 0 0 0 0.2 1.4 0.620:2(D11,14) 0.2 0 0.5 0.4 0.4 0.2 0.2 0.3 3.1 0.8 1.6 2.2 0 0.5 –0.1 0.5 1.2 0.6 3.3 0.720:3(D5,11,14) 0.1 0 2.8 1.3 0.2 0.4 0.6 0.5 0.6 2.6 14.6 6.7 0 0.3 1.3 0.7 –0.5 1.8 10.5 5.420:3(D11,14,17) 0 0 0 0 0 0 0 0 2.0 0.4 0.5 1.3 0 0 0 0 0 0 0 0SD5 1.1 1.1 35.2 12.8 6.0 6.9 74.2 31.7 0.6 2.9 16.1 7.3 0.4 1.6 56.8 18.2 1.6 2.9 31.0 14.1

Lipid Technology June 2013, Vol. 25, No. 6 137

butes to triacylglycerol-lowering properties such as, (i) decreasedde novo lipid synthesis, (ii) reduced substrate availability for lipo-protein formation, or (iii) changes in VLDL physicochemicalproperties. Therefore, PLA supplementation may reduce triacyl-glycerol and VLDL levels, suggesting a potential benefit in lower-ing high blood triacylglycerol levels [3].

Another health benefit of PLA is its appetite-suppressingeffect. PLA in the gastro-intestinal tract exerts this effect by trig-gering the release of the satiety gut hormones cholecystokinin(CCK) in the proximal small intestine (duodenum), and glucagonlike peptide-1 (GLP-1) in the distal small intestine (ileum). Theeffects of PLA on CCK response and appetite can be blocked bythe lipase inhibitor tetrahydrolipstatin. Therefore, the uptakeand metabolism of the precursor TAG to the free fatty acid formwhich is responsible for CCK and GLP-1 release, is needed toinduce satiety. In addition, The effect of PLA on intestinal hor-mone release is most likely mediated through chylomicron for-mation. Fatty acids with a chain length of more than 12 carbonatoms are absorbed into the circulation as chylomicrons. CCKsignaling pathways are closely related to the transport of chylo-microns. PLA affects chylomicron formation or transport andthereby influences release of CCK. This release leads to a delay ingastric emptying and in turn to an increased feeling of satietyand a decreased appetite. On the other hand, PLA induced GLP-1released from the distal small intestine may be of more impor-tant in suppressing post-meal intake by sustaining inter-mealsatiety. The relative contributions of these two hormones to theeffects of PLA on within-meal satiation, post-meal satiety andassociated changes in body weight over repeated dosing remainto be clarified [4].

Enrichment of PLAEnrichment of PLA can be carried out using either a physicalmethod or an enzymatic reaction. Urea complexation is a clas-sic method for fractionating fatty acids. Under certain condi-tions, urea forms a complex in spiral-shaped structures withhigh-carbon-number alkanes. Strong van der Waals attractionsexist between the urea molecules and high carbon-numberalkanes, which are held in circular channels of urea complex.Most urea complexes have hexagonal crystalline structurewhile pure urea crystals are tetragonal. Alkanoic acids, whichpossess long and saturated hydrocarbon chains, form ureainclusion complexes. In contrast, branched and cyclic mole-cules, or substances with chain length less than 6 to 8 carbonatoms, rarely form urea complexes. Also, fatty acids of shorterchain length or containing constituents such as double bonds,as well as epoxy or hydroxyl functional groups, are less likelyto form complexes. Depending on the choice of solvent and theratio of urea to fatty acid, inclusion of fatty acid in the ureacomplex can be controlled. As a solvent, methanol or ethanol isused traditionally. With higher urea to fatty acid ratios morefatty acids form a complex with urea and possibly, even someunsaturated fatty acid can form a urea inclusion complex. Theprevious study from our research group showed the effect ofurea to fatty acid ratio with a different solvent. As a result, thelevel of PLA was increased from 14.1 to 16.8, 23.0, and 45.1%with the ratio of urea to fatty acid, 1:1, 2:1 and 3:1 in ethanol,respectively. In methanol, PLA was enriched from 14.1 to 15.1,20.4, and 33.6%, with the ratio of urea to fatty acid, 1:1, 2:1 and3:1, respectively. The highest concentration of PLA (a 3.2-foldelevation) was achieved by crystallization with urea to fattyacid ratio, 3:1 in ethanol [5].

For enrichment of PLA, enzymatic reaction can be consideredvery effective because of the distinctive fatty acid distribution ofthis acid in the TAG molecule. As previously mentioned, D5-UPI-FAs, especially PLA, are almost exclusively esterified to the sn-3position of TAG from several seeds from conifer species. Immobi-lized lipase from Candida antarctica B (commercially Novozyme435) has a specificity toward fatty acids in sn-3 position of TAG inthe presence of excess ethanol (>3:1 (mol/mol), ethanol to TAG).In other words, when ethanolysis is carried out with TAG of pinenut oil, PLA primarily reacts with ethanol and produces fattyacid ethyl ester (FAEE) due to the specificity of immobilizedlipase from C. antarctica B. Thus, PLA is enriched in the FAEE veryefficiently with mild conditions. Our research group previouslystudied ethanolysis of pine nut oil with immobilized lipase fromC. antarctica B in a recirculated packed bed reactor. As a result,over 36% of PLA was enriched from initial 13% with the yield ofover 40% [6].

Enzymatic reaction for enrichment of fatty acid depends onthe specificity of lipases and on the properties of the substrateoil. Thus, choosing an appropriate lipase for a substrate is veryimportant. In pine nut oil, oleic acid and linoleic acid comprisea large portion of the fatty acids. For example, in case of Pinuskoraiensis, the total percentage of oleic acid and linoleic acid ismore than 70%. Both oleic acid and linoleic acid are D9-unsatu-rated fatty acid. Therefore eliminating these two fatty acidsfrom mixture of fatty acids of pine nut oil can contribute toenrichment of PLA. A lipase from Candida rugosa, which has spe-cificity toward conventional D9-unsaturated fatty acids, is idealenzyme for esterification reaction of fatty acid from pine nut oiland alcohol. Through the esterification reaction, in the presenceof Candida rugosa lipase, oleic acid and linoleic acid primarilyreact with alcohol and form fatty acid-alcohol esters. On theother hand, PLA, which is D5-unsaturated fatty acid, reactsmuch less readily and remains as an unreacted free fatty acid.Thus, it can be enriched in the unreacted fatty acid fraction. Ourresearch group recently studied an enzymatic esterification offatty acid from pine nut oil with lauryl alcohol using C. rugosalipase. Through the reaction PLA was enriched by up to 43 mol%from initial 13 mol% in the free fatty acid fraction [7].

Other attempts to improve PLA-containing oil

From our research group, several trials to improve PLA contain-ing oil have been explored. One of the attempts was synthesizinga monoacylglycerol containing PLA. Monoacylglycerols (MAG)are widely used as emulsifiers in the food, cosmetics, and phar-maceutical industries. MAG are also used in textile processing,in the production of plastics, and in the formulation of oils fordifferent types of machinery. A stepwise enzymatic reaction wascarried out with fatty acids from pine nut oil and glycerol usingcold active lipase from Penicillium camembertii as a biocatalyst.The reaction was first carried out the temperature at 208C andthen –108C. As a result, the maximum content of MAG of 88%was obtained after the two-step reaction [8].

As a subsequent trial, a structured lipid, which contains PLAin the sn-2 position of TAG and medium chain fatty acids (MCFA)in the sn-1,3 positions of TAG, was synthesized. Lipase-catalyzedacidolysis of modified pine nut oil with capric acid was carriedout in a continuous packed bed reactor (PBR) using LipozymeRM IM from Rhizomucor miehei as a biocatalyst. From a humannutritional point of view, structured lipid possessing a desiredfatty acid located at the sn-2 position with MCFA at the sn-1,3


138 June 2013, Vol. 25, No. 6 Lipid Technology

positions of TAG may have desirable characteristics becauseMCFA located at the end positions are rapidly hydrolyzed by pan-creatic lipases, readily absorbed in the intestines, and rapidlytransported to the liver, where they are metabolized as a quickenergy source. The remaining 2-MAG is well absorbed throughthe intestinal wall. Thus, synthesizing structured lipid contain-ing PLA in the sn-2 position of TAG can magnify the advanta-geous effects of PLA in the body [9].

In another attempt, lipase-catalyzed acidolysis of menhadenoil with a PLA concentrate, prepared from pine nut oil, was car-ried out in a solvent-free system. Incorporation of PLA from pinenut oil in fish oil TAG would provide unique specialty oil for spe-cific nutritional and clinical purposes. In the study, two differ-ent types of structured lipid were synthesized. The first type,which has PLA residues as the predominant fatty acid residue atthe sn-1,3 positions of the TAG, was synthesized using a 1,3-regiospecific lipase, namely, Lipozyme RM IM from Rhizomucormiehei. The second type of SL containing PLA residues as the pre-dominant fatty acid residue at both the sn-1,3 and sn-2 positionswas produced with Novozym 435 from Candida antarctica [10].

Conclusion

Due to increasing number of vegan/vegetarian population in theworld, the need for finding a plant source polyunsaturated fattyacid is on the rise. In addition, off-flavor of traditional polyunsa-

turated fatty acid from marine source adversely affects consu-mers' taste. PLA is an emerging health-benefiting source ofplant-oriented polyunsaturated fatty acid. It has distinctive char-acteristics and several health-promoting effects, coming from itsD5-unsaturation. Several studies concerned with the enrich-ment of PLA from pine nut oil using physical and enzymaticmethod along with utilizing PLA to form other lipid productshave been conducted. Even though all the evidence shows itspotential to be a new position as more important and wide-spread source of polyunsaturated fatty acid, PLA is relativelyunfamiliar to majority of population.

References

[1] Wolff, R.L. et al., Lipids 2000, 35, 1 –22.

[2] Wolff, R.L. et al., J. Am. Oil. Chem. Soc. 1997, 74, 515–523.

[3] Asset, G. et al., Lipids 1999, 34, 39–44.

[4] Hughes, G.M. et al., Lipids Health Dis. 2008, 7, 1 –10.

[5] Lee, J.-W. et al., Lipids 2004, 39, 383 –387.

[6] Zhao, T. et al., J. Food Sci. 2012, 77, C267 –C271.

[7] No, D.S. et al., 104th AOCS Annual Meeting & Expo., 2013.

[8] Pyo, Y.-G. et al., Biotechnol. Progr., 2012, 28, 1218–1224.

[9] Choi, J.-H. et al., J. Am. Oil. Chem. Soc. 2012, 89, 1449–1454.

[10] Kim, I.-H. et al., J. Am. Oil. Chem. Soc. 2006, 83, 109–115.


pinolenic acid as a new source of phyto-polyunsaturated fatty acid

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