Policosanol characterization and accumulation during ripening of Tunisian Olea europaea L. fruits

Research Article

Policosanol characterization and accumulation duringripening of Tunisian Olea europaea L. fruits

Faouzi Sakouhi1, Sadok Boukhchina1, Christelle Absalon2, Eric Fouquet2 and Habib Kallel1

1 Laboratoire de Biochimie des Lipides, Département de Biologie, Faculté des Sciences de Tunis, El Manar,Tunisie

2 Centre d’Etude Structurale et d’Analyse des Molécules Organiques, Institut des Sciences Moléculaires, Uni-versité de Bordeaux, Bordeaux, France

Policosanol is a mixture of bioactive molecules shown to have beneficial effects in treating hypercholes-terolemia. Food products enriched in policosanol are currently available in the US market. In the presentstudy, eight policosanol components were identified by GC-MS during the ripening of Meski olives. Thequantitative characterization of these compounds was performed using GC-FID. The results showed thatthe maximum level of total policosanol components (947.20 mg/100 g oil) was reached at the 26th weekafter the flowering date of Meski olives. Hexacosanol and tetracosanol were the predominant policosanolcomponents at Meski olive maturity. However pentacosanol, heptacosanol and tricosanol were less presentin the olives and they accounted for 14% of the total policosanol at complete maturity of the fruit. The totalpolicosanol content of Meski olives was higher than that of beeswax and whole sugar cane, which belong tothe sources of dietary supplements containing policosanol. These findings indicate that olive is a potentialsource of these health-enhancing compounds for functional foods and nutraceutical applications.

Keywords: Accumulation / Characterization / Olive oil / Policosanol / Ripening

Received: April, 6 2009; accepted: July, 7 2009

DOI 10.1002/ejlt.200900076

Eur. J. Lipid Sci. Technol. 2010, 112, 373–379 373

1 Introduction

Policosanol is a group of natural products present in theunsaponifiable fraction of vegetable oils [1] and is veryimportant in the human diet [2, 3]. It is a common termthat refers to a mixture of long-chain (20–36 carbons) ali-phatic primary alcohols. The mixture contains mainly doc-osanol, tetracosanol, hexacosanol, octacosanol and tria-contanol [4]. These compounds have been widely studiedfor their health properties [5–7]. The unsaponifiable fractionof vegetable oils contains several compounds of sterols,phytostanols, triterpenic acids, tocopherols and long-chainaliphatic alcohols (policosanol) whose biological activitiesare important [8–12]. Policosanol was originally isolatedfrom sugar cane, beeswax, rice bran and wheat germ [13,

14]. It is also present in fruit, seeds, leaves and on surfacesof plants [4]. Numerous beneficial physiological effects havebeen attributed to policosanol [6], such as reducing plateletaggregation and endothelial damage, and cholesterol-low-ering effects [15–17]. In addition, several studies reportedthe beneficial health effects for each individual policosanolcompound. Octacosanol is in fact known for its anti-aggre-gant effect, as an alternative to aspirin for patients sufferingfrom gastric irritation, due to its cytoprotective effect [17].Triacontanol has, however, anti-inflammatory activities andit controls the chemical composition and physical status ofmembrane lipids [18, 19]. Owing to policosanol’s importanthealth effects, a target of recent researches is the isolation ofnatural aliphatic alcohols of the series C24–C34 which, oncepurified, have medical appellation (Policosanol™) [20].Thus, the industrial objective is to identify plant matricesrich in these beneficial health compounds. Currently, anumber of dietary supplements containing policosanol arecommercially available in the US market [4]. These supple-ments are mainly prepared from beeswax or sugar caneextracts. However, the olive policosanol components havereceived insufficient attention. In fact, olive products (oil

Correspondence: Faouzi Sakouhi, Laboratoire de Biochimie desLipides, Département de Biologie, Faculté des Sciences de Tunis, 2092El Manar II, Tunisie.Fax: 1216-7-1885480E-mail: [email protected]

© 2010 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim www.ejlst.com

374 F. Sakouhi et al. Eur. J. Lipid Sci. Technol. 2010, 112, 373–379

and table olives) are well-known sources of healthy micro-nutrients and are their main dietary source in the Medi-terranean countries. Tunisian annual production is about210,000 t of oils and 23,000 t of table olives [21].

Although the policosanol contents of beeswax, sugar caneand wheat have been extensively studied [4, 22], very little re-search information is available about olive policosanol char-acterization. Therefore, the goal of our investigation is todetermine the policosanol content and composition during theripening of Meski olives (Tunisia), and then to determineexactly the time when the olive accumulates maximally thesebeneficial health compounds. A comparative policosanolcharacterization between olive, beeswax, sugar cane andwheat has also been established.

2 Material and methods

2.1 Plant material

The Meski variety of olive (Olea europea L.) was grown on theAgronomy farm of the O.T.D.G. (Office Terres DomanialesGhzala, Bizerte) in the north of Tunisia. Olives were hand-harvested from the same tree at intervals of 1 week, from theformation of the olive [21st week after the flowering date(WAF) of Meski olives] until their complete maturity (38th

WAF). Only healthy fruits, without any sign of infection orphysical damage, were selected.

2.2 Reagents and standard

Acetone, chloroform, diethyl ether and petroleum ether werepurchased from Fisher Scientific (Loughborough, UK).Ethanol was from Scientific Limited (Northampton, UK).Pure aliphatic alcohols, 1-eicosanol [internal standard (IS)]and N,O-bistrimethylsilyltrifluoroacetamide (BSTFA) wereacquired from Sigma (St. Louis, MO, USA). TLC silicaplates (silica gel 60 G F254, 20620 cm, 0.25 mm thickness),potassium hydroxide pellets and anhydrous sodium sulfatewere from Merck (Darmstadt, Germany). Pyridine was pur-chased from Fluka (Neu-Ulm, Germany).

2.3 Determination of oil content

The oil content was determined by extracting dry material ofolives (Olives were dried at 20 7C in dry-air sterilizers) withpetroleum ether, using a Soxhlet apparatus [23]. The extrac-tion was performed for 4 h at 42 7C and was repeated threetimes for each sample. The extract was dried in a rotary eva-porator at 32 7C. Oil was weighed and stored at –10 7C. TheSoxhlet extraction method of lipids was characterized by itshigher yield of oil compared to the mechanical ones, press orcentrifugation, especially at early stages of olive developmentwhen the fruit was immature [24].

2.4 Saponification

Oil samples were saponified following the method describedby Sakouhi [12]. In brief, the unsaponifable fraction of lipidswas determined by saponifying 5 g of oil mixed with both100 mL 1-eicosanol solution (0.1% wt/vol) and an ethanolicKOH 12% wt/vol solution; the mixture was heated at 60 7C for1.30 h. After cooling, 50 mL H2O was added. The unsaponi-fiable matter was extracted four times with 50 mL petroleumether. The combined ether extract was washed with 50 mLethanol/water (1 : 1 vol/vol). The extracted ether was driedover anhydrous Na2SO4 and evaporated to dryness using N2.The dry residue was dissolved in chloroform for TLC analy-sis.

2.5 Thin-layer chromatography

The separation of the unsaponifiable fraction by TLC wascarried out according to Irmak [4] after slight modification bySakouhi [12]. Briefly, the unsaponifiable matter was separatedinto subfractions on preparative silica gel thin-layer plates(silica gel 60 G F254) using one-dimensional TLC withhexane/diethyl ether (6 : 4 vol/vol) as the developing solvent.The unsaponifiable fraction diluted in chloroform was appliedto the silica gel plates. To correctly identify the sterols band, areference sample (1-eicosanol) was applied on the left and theright sides of the TLC plates. After development, the platewas sprayed with 2,7-dichlorofluorescein and viewed underUV light. The band corresponding to aliphatic alcohols wasscraped off, extracted three times with chloroform/diethylether (1 : 1 vol/vol), filtered to remove the residual silica, driedin a rotary evaporator and stored at –10 7C.

2.6 Silylation of aliphatic alcohol fraction

An amount of 2 mg of aliphatic alcohol residue was mixedwith 125 mL BSTFA (with 1% trimethylchlorosilane), 125 mLpyridine and 450 mL acetone. The mixture was vortexed forabout 10 s and heated at 70 7C for 20 min. After the silylationreaction, 1.5 mL chloroform was added to the mixture and1 mL of the solution was directly injected into the gas chro-matograph.

2.7 Gas chromatography conditions

Aliphatic alcohol compounds were analyzed using a DB-5MSfused-silica capillary column (30 m60.25 mm I.D., 0.25 mmfilm thickness; J&W Scientific, Folsom, CA, USA) in a VarianSAR 3400Cx gas chromatograph coupled directly to the massdetector (MS Varian SATURN). Helium was used as carriergas, with a constant flow rate of 1 mL/min. The injector anddetector temperatures were 250 7C. The oven temperaturewas programmed from 150 to 300 7C at 4 7C/min. The finaltemperature was held constant for 10 min and the transfer linetemperature was 250 7C. Electron impact mass spectra were


Eur. J. Lipid Sci. Technol. 2010, 112, 373–379 Policosanol accumulation in olives 375

measured at an acceleration energy of 70 eV. Manual injectionof 1 mL of the sterol solution was performed in the split modeat a 60 : 1 split ratio. The aliphatic alcohols were identified bycomparing their retention times and mass spectra with thoseof their pure molecules. The peaks were also confirmed withthe NIST Mass Spectral Library. The retention time and massspectrometric data of policosanol compounds identified byGC-MS are provided in Table 1.

Due to the higher sensibility of the GC-FID detectorcompared to the GC-MS one [12, 25], the quantification ofpolicosanol was performed using GC-FID. The GC systemused was a HP 4890A gas chromatograph (Agilent Technol-ogies, Palo Alto, CA, USA) equipped with a split-splitlessinjector and an FID, and a DB-5MS (30 m60.25 mm I.D.,0.25 mm film thickness; J&W Scientific, Folsom, CA, USA)column was used. The analyses were performed using thesame chromatographic conditions as for GC-MS. Quantifi-cation of policosanol was achieved by addition of 1-eicosanolas internal standard. The level of each policosanol componentwas calculated as mg/100 g of oil using the following formula:amount = 100 – (PAs)(mis)/(PAis)(m), where PAs is the poli-cosanol peak area, PAis is the internal standard area, mis is theweight (mg) of the internal standard, and m is the weight (g) ofthe oil taken for analysis. The policosanol content (expressedin mg/100 g of oil) was determined for three independentreplicates at each stage of olive maturity.

3 Results and discussion

3.1 Policosanol characterization at complete maturityof olives

The results from the qualitative characterization of Meski fruitusing GC-MS indicated that the aliphatic alcohol fraction ofthe olives was made up of very-long-chain aliphatic primaryalcohols (Table 1). Moreover, the results showed that all thecompounds of the aliphatic alcohol fraction belonged to the

policosanol group (aliphatic alcohols with 20–36 carbons).The identified components were divided into two groups: thepaired policosanol carbon chain (PPCC) group representedby docosanol (C22H45OH), tetracosanol (C24H49OH), hexa-cosanol (C26H53OH), octacosanol (C28H57OH), and tria-contanol (C30H61OH), and the impaired policosanol carbonchain (IPCC) group represented by tricosanol (C23H47OH),pentacosanol (C25H51OH), and heptacosanol (C27H55OH).

The quantitative characterization of policosanol (ex-pressed in mg/100 g of oil), at complete maturity of the Meskiolives, showed that hexacosanol (70.12 mg/100 g oil), tetra-cosanol (48.30 mg/100 g oil), octacosanol (35 mg/100 g oil)and docosanol (25.62 mg/100 g oil) were the dominant poli-cosanol compounds accounting for over 85% of the totalpolicosanol. However, pentacosanol (11.96 mg/100 g oil),heptacosanol (10.15 mg/100 g oil) and tricosanol (4 mg/100 g oil) were less present in Meski olives. Moreover, theresults from the quantitative characterization of samplesexamined here showed that, at the stage of complete maturity,the PPCC compounds had accumulated to higher amountsthan the IPCC compounds in the Meski olive cultivar. Thequantitative characterization of policosanol at completematurity of our samples was in agreement with that reportedby Fernández-Arche et al. [26]. Indeed, these authors haveperformed policosanol characterization only at completematurity of the fruit and they demonstrated that Spanish oliveoils contained mainly hexacosanol (37.3%), tetracosanol(30.5%) and octacosanol (15.3%). Additionally, these authorsmentioned that IPCC were less present in Spanish oliveproducts, accounting for 4.5% of the total policosanol. Octa-cosanol was one of the major PPCC (35 mg/100 g oil) inMeski olives; it is in fact known for its anti-aggregant effect, asan alternative to aspirin for patients suffering from gastricirritation, due to its cytoprotective effects [17]. Moreover,triacontanol, a natural compound having anti-inflammatoryand membrane-stabilizing effects [18, 19], was also present(11 mg/100 g oil) in Meski olives. Thus, the high PPCC levelcontributes to the great beneficial effects of olives, as reported

Table 1. Retention time and mass spectrometric data for aliphatic alcohol components identified by GC-MS.

Retentiontime [min]

Main fragmentation ions, m/z Compound Chemical formula

10.56 252, 167, 139, 111, 83, 43 Eicosanol (IS) C20H42O13.32 280, 195, 111, 69, 55, 43 Docosanol C22H46O16.64 308, 223, 125, 111, 57, 43 Tricosanol C23H48O19.10 336, 252, 153, 111, 97, 57 Tetracosanol C24H50O21.95 350, 265, 139, 97, 83, 43 Pentacosanol C25H52O25.08 364, 236, 125, 111, 97, 57 Hexacosanol C26H54O28.11 378, 208, 111, 97, 71, 29 Heptacosanol C27H56O31.09 364, 265, 125, 97, 69, 43 Octacosanol C28H58O36.65 392, 336, 223, 139, 111, 57 Triacontanol C30H62O

IS, Internal standard.



in recently published studies focusing on its healthy effects[18, 19, 27].

All the studies showed that sugar cane and beeswax are themajor sources of dietary supplements containing policosanol[4, 20, 22]. In fact, the total policosanol levels of whole sugarcane, sugar cane leaves and sugar cane peel were 17.4, 181and 270 mg/kg, respectively. However, the correspondingfigures for beeswax-brown and beeswax-yellow were 5.2 and12 mg/kg, respectively. Moreover, the total policosanol con-tent of the samples examined here was 20.5 mg/kg. Theseresults showed that the total policosanol content of Meskiolives was higher than that of beeswax (brown and yellow) andwhole sugar cane. This comparison of policosanol contentindicates that Meski olives may be a potential source of poli-cosanol.

Commercial policosanol-enriched products have numer-ous matrix sources (sugar cane, beeswax, wheat, etc.). In fact,sugar cane wax contained mostly octacosanol (60–70%) fol-lowed by hexacosanol, triacontanol and dotriacontanol [28].In wheat bran, the predominant policosanol components weretetracosanol (35%), hexacosanol (15%) and octacosanol(12%) [4]. In beeswax, Michael et al. [22] reported that themain policosanol compounds were triacontanol (36.9%),dotriacontanol (20.8%), octacosanol (18.3%), hexacosanol(13.9%) and tetracosanol (9%). However, our results showthat Meski olives contained hexacosanol (34.12%), tetra-cosanol (23.50%), octacosanol (17.03%) and docosanol(12.46%). These results show that the individual compositionof policosanol compounds is source dependent. Thus, thequalitative and quantitative characterization of policosanolcompounds may be used as marker to check the matrix originof policosanol-enriched products.

3.2 Change in policosanol content during maturation

It is interesting to mention that the change in policosanolcontent during olive development has never previously beenstudied. The results from the quantitative characterization ofsamples examined here showed that, from the 21st WAF, thelevel of hexacosanol, a major compound of policosanol, start-ed to increase gradually and reached its maximum (340.42 g/100 g oil) at the 26th WAF of the Meski fruit (Fig. 1), but adramatic decrease in the amount of this compound wasobserved from the 27th WAF, to attain its lowest level at com-plete maturity of the Meski olives (70.12 mg/100 g oil). Fig-ures 1 and 2 show that the patterns of PPCC compounds weresimilar during the ripening of Meski olives. This similaritycould probably be explained by the fact that these compoundshad the same biosynthetic precursor. Moreover, the resultsindicated that hexacosanol and triacontanol were the majorand the minor PPCC compounds, respectively, during theripening of Meski olives. The maximum level of PPCC wasattained at the 26th WAF of olives and was 233.72, 170, 127.5and 34.94 mg/100g oil for tetracosanol, octacosanol, doc-osanol and triacontanol, respectively. Consequently, thehigher amount of paired policosanol explains the nutritionalbenefits of the olives, since numerous researches indicated thebeneficial health effects of octacosanol and triacontanol [17,29]. At the 27th WAF, a great decrease in PPCC compoundaccumulation was observed. The decline in these policosanolcomponents may be due to their conversion to others me-tabolites such as fatty acids [30]. In fact, Menéndez et al. [31]demonstrated that shorter (myristic, palmitic and stearic) andunsaturated (oleic and palmitoleic) fatty acids are formed afteroral dosing of monkeys with policosanol. Consequently, the

Figure 1. Changes in total policosanol (1), hexacosanol (m) and tetracosanol (s) contents during ripening ofMeski olives.



Figure 2. Variation of octacosanol (d), docosanol (m) and triacontanol (s) amounts during ripening of Meskiolives.

results from the quantitative characterization of policosanolduring the ripening of olives have a great importance; in fact,in Meski fruit, the highest amount of PPCC was reached at the26th WAF. At this time, the total policosanol content was aboutfive times higher than that determined at complete maturity ofthe Meski fruit. Therefore, at this stage, the Meski olive maybe a potential source of these health-enhancing compoundsfor functional foods and nutraceutical applications.

Concerning IPCC, our results showed that these com-pounds were found to be minor components of the aliphaticalcohol fraction. This group contained, in decreasing con-centration, pentacosanol, heptacosanol and tricosanol. Fig-ure 3 shows that the greatest change in IPCC content oc-curred during the early stages of olive development, in con-trast to PPCC. Therefore, the maximum level of these alcoholcomponents was reached at the 21st WAF and was 32.15,29.81 and 22.31 mg/100 g oil for pentacosanol, heptacosanoland tricosanol, respectively. During the ripening of Meskiolives, pentacosanol and heptacosanol accumulated nearly inequal proportions. The total IPCC proportion changed from5 to 16% of the total aliphatic alcohol fraction during thedevelopment of the Meski olives. Figure 4 shows that thegreatest change in IPCC and PPCC occurred during theripening of the Meski olives. The highest percentage of IPCCwas observed early at the 22nd WAF (15.78% of total polico-sanol), while the highest PPCC was attained at the 26th WAF(95.76% of total policosanol). These results showed that,during the ripening process, the olives modified the biosyn-thesis of PPCC and IPCC according to their need for thesecompounds to achieve their physiologic functions, such as theconversion of PPCC to the biosynthesis of fatty acids via b-oxidation [31]. It will be of great interest to mention thatrecent clinical studies demonstrate that the average dietary

consumption of policosanol ranges from 2 to 20 mg per day.Moreover, other studies attest that the intake of policosanolranging from 5 to 10 mg per day leads to a 15–30% reductionof LDL-cholesterol in humans [28, 32]. Furthermore, inhumans, the average dietary consumption of policosanol isclosely dependent on sex and age [33–35].

In conclusion, the present study results show that olivescontain an interesting quantity of policosanol and that theycan be a significant dietary source of policosanol-enrichedproducts. The total policosanol content of olives is higher thanthat of beeswax (brown and yellow) and whole sugar cane.PPCC are present in higher amounts than IPCC at completematurity of the Meski olives, which contributes to the greatbeneficial effects of olive products. The maximum accumula-tion of policosanol was observed at the 26th WAF, which is thebest time to maximally exploit these beneficial health compo-nents. Thus, at the 26th WAF, Meski olives may be a potentialsource of natural bioactive compounds for incorporation intofunctional foods and nutraceuticals.

Acknowledgments

This work was done as part of a National Research Project.We thank the Ministry of Scientific Research, Technology andCompetence Development of Tunisia for financially support-ing this investigation. Part of this work was carried out at theCentre d’Etude Structurale et d’Analyse des Molécules Orga-niques (CESAMO), Bordeaux, France.

Conflict of interest statement

The authors have declared no conflict of interest.



Figure 3. Changes in impaired policosanol contents during ripening of Meski olives: pentacosanol (d), hep-tacosanol (m) and tricosanol (s).

Figure 4. Variation of paired policosa-nol (PPCC - diagonal hatching) andimpaired policosanol (IPCC - dashed)proportions during ripening of Meskiolives.

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Policosanol characterization and accumulation during ripening of Tunisian Olea europaea L. fruits