72 0009-3130/14/5001-0072 ©2014 Springer Science+Business Media New York

Chemistry of Natural Compounds, Vol. 50, No. 1, March, 2014 [Russian original No. 1, January–February, 2014]

LIPIDS FROM Lycopersicon esculentum FRUIT SKIN

N. T. Ul�chenko,* S. D. Gusakova, and Sh. K. Khidoyatova

Free (FL) (1.25 and 1.22%) and bound lipids (BL) (1.75 and 1.63%) were isolated from skin of ripeLycopersicon esculentum fruit grown in open plots and in a hothouse. The groups and classes of lipids wereestablished. The FL consisted of 9.3% natural fatty acid ethyl esters and 3.7% carotinoid pigments. Thecompositions of the hydrocarbons, aliphatic and cyclic alcohols, and fatty acids of all lipid classes weredetermined. Saturated fatty acids, mainly 16:0, dominated the majority of acyl-containing lipids. Acid 18:2dominated the unsaturated acids.

Keywords: Lycopersicon esculentum, tomato, fruit skin, lipids, carotinoids, fatty acids, fatty acid ethyl esters.

Lycopersicon esculentum (Tourn.) Mill. (Solanaceae) is a widely distributed vegetable crop. The chemical compositionof the fruit is rather well studied. Sugars, pectinic substances, purines, organic acids (tanacetic, malic, citric, etc.),dihydroxystearic acid, alkaloids (clavatin, clavotoxin, nicotine, etc.), carotinoids, and vitamins have been found in them [1].Tomato seeds contain up to 30% semi-drying oil, which possesses high gustatory qualities and has food and biological value[2, 3].

Skin of tomato fruit yielded the red carotinoid pigment lycopene, which has high antioxidant activity and radioprotective,hypolipidemic, and antiproliferative properties [4].

Data on the lipid composition of tomato skin are limited. It was reported that skin of Egyptian tomatoes afforded 4%lipids whereas their fatty acid (FA) composition had unsaturated FA (77.6%) with 18:2 acid predominating [5]. A broaderspectrum of FAs including the usual components of the isologous series 16:1-16:4, isomers of 18:1 (n-9, n-11) and 20:4(n-3, n-6), and acids 18:4 and 22:5 (n-3) was found in the lipid extract obtained from the skin of Spanish tomatoes by CO2extraction [6].

We studied lipids from skin of ripe tomato fruit grown on open plots (I) and in hothouses (II) in Uzbekistan.Skin separated from fruit was dried in air to residual moisture content 6.5% (I) and 6.8 (II) and ground. Free lipids

(FL) were isolated from the skin powder using extraction hydrocarbons; bound lipids (BL), a CHCl3:MeOH mixture. Thecontents of carotinoid pigments in the FL, unsaponified substances (US) isolated from the basic hydrolysis products of the FL,and carotinoids in the US were determined. The content of pigments in the BL was determined first. Then, they wereseparated by column chromatography (CC) over silica gel into neutral lipids (NL) (hydrocarbon eluent), glycolipids (GL)(CHCl3), and phospholipids (PL) (MeOH). Table 1 presents the results.

Table 1 shows that the contents in skin of the two samples of FL and BL in addition to carotinoids and US in FL of Iand II were practically identical. The US in I and II comprised greater than 30% of the lipid mass; the content of carotinoidsin US, 9–9.3%.

However, the quantitative group compositions of BL of I and II differed considerably. NL predominated by mass inBL of I; GL, in BL of II. The fraction of PL in BL of II was twice that in BL of I. GL were separated into separate classes byTLC on silica gel using solvent system 1; PL, systems 2 and 3. Literature data, specific detectors, and model compounds wereused to identify the classes of GL and PL [7, 8]. GL of both samples contained sterylglycosides and their fatty-acid esters,mono- and digalactosyldiacylglycerides, and cerebrosides. The dominant constituents of GL were sterylglycosides.Phosphatidylcholines (basic class), phosphatidylethanolamines, and phosphatidylinositols were identified in the PL.

S. Yu. Yunusov Institute of the Chemistry of Plant Substances, Academy of Sciences of the Republic of Uzbekistan,100170, Tashkent, e-mail: natalya0150@mail.ru. Translated from Khimiya Prirodnykh Soedinenii, No. 1, January–February,2014, pp. 67–70. Original article submitted October 7, 2013.

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The compositions and contents in FL of I and II were established using tomato-skin FL I as an example because of thesimilarity of their principal parameters. FL were separated by preparative TLC (PTLC) on silica gel using solvent system 4into separate classes. The fraction consisting of free FA (FFA), aliphatic alcohols, and triterpenols was treated with diazomethaneto convert the FFA into the methyl esters and then rechromatographed by PTLC using solvent system 5. The contents of thelipid classes were established gravimetrically. Table 2 presents the results.

The principal components of the identified FL classes were hydrocarbons and FFA. This differentiated them fromseed lipids, where triacylglycerides (TAG) dominated [2]. The fraction of FA esters (FAE) with aliphatic and cyclic alcoholswas practically the same as that of TAG. Free cyclic alcohols (sterols and triterpenols) together with FAE made up almost afifth of the FL mass.

The hydrocarbon fraction according to TLC using solvent system 6 included alkanes with alkenes (Rf 0.96, detectionby I2 vapor), �-carotene (Rf 0.54), and lycopene (0.27). According to photoelectrocolorimetry, the lycopene content was96.5% of the carotinoid mass; �-carotene, 3.5%.

The compositions of hydrocarbons, aliphatic alcohols, sterols, and triterpenols were established by GC using compoundsisolated from natural sources as models [9].

Hydrocarbons were according to GC mainly alkanes consisting of 19 homologs of even and odd series with chainlengths 14:0–34:0 and were dominated by odd homologs (85.67%), the principal ones being 31:0 (50.6%) and 29:0 (20.64%)(Table 3). Clear peaks of alkenes were missing in the GC chromatogram. Therefore, their contents were not determined.

Aliphatic alcohols were represented by 11 constituents with docosanol (22:0), tricosanol (23:0), and tetracosanol(24:0) dominating (Table 3).

TABLE 1. Lipid Content in Fruit Skin of Open-Plot (I) and Hothouse (II) Tomatoes, %

Parameter ² ²² Parameter ² ²²

Free lipids including: carotinoids, % of FL mass unsaponified substances carotinoids, % of US mass

1.25 3.70 0.40 9.00

1.22 3.16 0.38 9.33

Bound lipids including: carotinoids, xanthophylls, % of BL mass neutral lipids glycolipids phospholipids

1.75 0.25 0.85 0.72 0.18

1.63 0.28 0.25 0.99 0.39

TABLE 2. Composition and Content of Free Lipid Classes of Tomato Fruit Skin

Neutral lipids Content, % of FL mass Neutral lipids Content, %

of FL mass

Hydrocarbons including carotinoids Fatty acid esters with aliphatic and cyclic alcohols Fatty acid ethyl esters Triacylglycerides Free fatty acids

19.5 7.5 9.3 7.3 21.5

Aliphatic alcohols Triterpenols Sterols Unidentified constituents Xanthophylls

5.1 6.5 4.6 18.6 0.1

TABLE 3. Composition and Content of Alkane Hydrocarbons and Fatty Alcohols of Free Lipids of Tomato Fruit Skin Accordingto GC

Chain length

Hydrocarbons, % of their mass

Aliphatic alcohols, % of their mass

Chain length

Hydrocarbons, % of their mass

Aliphatic alcohols, % of their mass

14:0 15:0 16:0 17:0 18:0 20:0 22:0 23:0 24:0 25:0

Tr. Tr. Tr. Tr. Tr. Tr.

0.28 0.52 0.18 0.45

– –

2.7 –

2.7 7.0 22.2 20.0 16.8 10.0

26:0 27:0 28:0 29:0 30:0 31:0 32:0 33:0 34:0

0.26 1.51 1.03 20.64 4.18 50.60 7.28 11.95 1.12

10.9 3.5 4.2 –

Tr. – – – –

______Tr. - traces.

74

Triterpene alcohols comprised mainly �-amyrin (72.6%) and �-amyrin (27.4%). Phytosterols included �-sitosterol(93.2%), stigmasterol (6.8%), and traces of campesterol.

Ethyl esters of FA (EEFA), which are a rarely encountered class of natural lipids and were detected earlier in surfacelipids of several higher plants and in seed lipids of seabuckthorn and Onopordum acanthium with contents <2.0% [10–12],were noted in the FL of tomato skin. The EEFA content in tomato-skin lipids was 9.3%. Their composition was established byGC. The models for identifying them were EEFA of sunflower oil, which we obtained by re-esterification of triacylglyceridesusing NaOEt [13]. The retention times of the EEFA for chromatography over a capillary column were 11.19 (14:0), 15.77(16:0), 20.155 (18:0), 24.235 (20:0), 15.047 (16:1), 19.608 (18:1 + 18:3), and 19.49 (18:2). These values differed considerablyfrom those of the FA methyl esters that were obtained under the same conditions [14]. The retention times did not differ ifpacked columns were used [11].

FA from other acyl-containing lipid classes were isolated after basic hydrolysis. The resulting FA were methylated bydiazomethane and analyzed by GC as the methyl esters (Table 4). FA were identified by comparing their retention times withthose of known constituents obtained under identical GC conditions [14].

Table 4 shows that the set of FA from tomato-skin lipids was rather varied and included saturated constituents primarilyof the even series 10:0–24:0, which were present in the total composition only in the FFA fraction. The greatest amount ofsaturated acids was found in the FAE and GL fractions with contents of about 65 and 70%, respectively. The EEFA fractionwas less saturated. The principal saturated acid with respect to content in all lipid classes was palmitic (16:0), from 23.6% inEEFA to 55.0% in GL. Unsaturated FA were represented mainly by three constituents, i.e., oleic (18:1), linoleic (18:2), andlinolenic (18:3) acids. The fraction of 18:2 acid was greatest in EEFA (up to 41.5%). The contents of 18:2 acid wereapproximately the same in TAG and PL.

Thus, FL and BL of tomato fruit skin were studied for the first time. It was shown that the skin was rich in not onlythe carotinoid pigment lycopene but also a complicated complex of biologically active lipids.

EXPERIMENTAL

GC of methyl esters was performed on an Agilent 6890N instrument with a flame-ionization detector according to theFAMES.M method using a capillary column (30 m � 0.32 mm) with HP-5 stationary phase, He carrier gas, and temperatureprogrammed from 150 to 270°C and on a Chrom-5 instrument with a flame-ionization detector using a steel column(2.5 m � 4 mm) packed with Chromaton-N-AW with 15% Reoplex-400, column temperature 192°C, and N2 carrier gas.GC of hydrocarbons and aliphatic and cyclic alcohols used a glass column (1.2 m � 3 mm) filled with Inerton-Super with 5%OV-1 and column temperature 220–260°C.

TABLE 4. Fatty Acid Compositions of Tomato Fruit Skin Lipid Classes, GC, % of Acid Mass

Fatty acid FL* FAE EEFA TAG FFA GL PL

10:0 12:0 14:0 15:0 16:0 16:1 17:0 18:0

18:1 + 18:3

18:2 20:0 20:1 22:0 24:0 �sat.

�unsat.

– 0.57 3.79 0.32

34.75 1.33 0.25 6.20

13.00 10.64 29.15

Tr. – – –

45.88 54.12

– 1.25 6.48 1.99

40.39 –

1.35 9.85

19.45

16.08 1.60

1.56

64.47 35.53

– –

1.18 –

23.65 0.77

– 7.16 25.26

41.50 0.48

– – –

32.47 67.53

– 0.89 2.32

– 33.87

– –

9.78 25.18

27.96

– – – –

46.86 53.14

0.33 0.51 2.14 0.60 43.81 2.69 0.51 6.18 19.76

22.21 0.51 0.28 0.23 0.24 55.06 44.94

– –

1.92 –

55.03 – –

11.35 12.72

17.57 1.41

– – –

69.71 30.29

– 0.30 1.34 0.46

45.88 0.44 0.81 7.90

15.36

26.97 0.54

– – –

57.23 42.77

75

The carotinoid content was determined on a KFK-2-UKhL 4.2 photoelectrocolorimeter as before [15].TLC of lipids was carried out on Chemapol (Czechoslovakia) silica gel of particle size 5/40 �m; CC, over silica gel

with particle size 100/160 �m.Solvent systems were CHCl3:Me2CO:MeOH:AcOH:H2O (65:20:10:10:3, 1); CHCl3:MeOH:NH4OH (13:7:1, 2);

CHCl3:MeOH:AcOH:H2O (14:5:1:1, 3); hexane:Et2O (4:1, 4; 3:2, 5); and heptane:C6H6 (9:1, 6). Spots of NL were detectedby I2 vapor and by spraying plates with aqueous H2SO4 (50%) with subsequent heating; GL, by �-naphthol; PL, by Vaskovskyand Dragendorff’s reagents.

Isolation of Lipids. FL were extracted from air-dried ground skin by standing five times with extraction hydrocarbons(bp 72–80°C). BL were extracted from the pulp also by standing five times with CHCl3:MeOH (2:1). The CHCl3:MeOHmixture was worked up with aqueous CaCl2 solution (0.05%) to remove non-lipid constituents. The yield of lipids wasestablished gravimetrically after distilling the extractants and subsequent drying of the residues in a vacuum drying cabinet at60°C. FA were obtained by basic hydrolysis of acyl-containing lipid groups [16]. Methyl esters were produced from FA usingfreshly prepared diazomethane [17]. US were isolated from the lipids as before [18].

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