ORIGINAL PAPER

Content and Fatty Acid Composition of Neutral Acylglycerolsin Euonymus Fruits

Roman A. Sidorov • Anatoly V. Zhukov •

Vasily P. Pchelkin • Andrei G. Vereshchagin •

Vladimir D. Tsydendambaev

Received: 10 June 2013 / Revised: 21 January 2014 / Accepted: 23 January 2014

� AOCS 2014

Abstract The content and fatty acid (FA) composition of

FA neutral acylglycerols (NAG), a mixture of 1,2,3-triacyl-

sn-glycerols (TAG) and 3-acetyl-1,2-diacyl-sn-glycerols

(acDAG), were determined in the seeds and arils of fruits

of 14 Euonymus L. species. On the average, the seeds

exceeded the arils in the absolute and relative dry matter

content 2.9- and 1.9-fold, respectively, and separate plant

species greatly differed in NAG composition. The pro-

portions of TAG in the NAG of seeds and arils were 4–5

and *98 %, respectively. The degree of FA unsaturation

in aril NAG was higher than in the seed NAG, and in

acDAG—higher, than in TAG. In the NAG, 14 major FA

molecular species (excluding minor FA) were found, and

linoleic, oleic, palmitic, and linolenic acids were predom-

inant. NAG of separate taxonomic units of the genus

Euonymus L. differed from each other in the concentration

of major FA as well as other FA. So, by using statistical

analysis, it was definitely established that the species from

the subgenus Euonymus were characterized by an increased

content of linoleic acid, while those from the subgenus

Kalonymus, by the predominance of oleic acid. Meanwhile,

the species of the section Euonymus were marked by an

enhanced concentration of a number of hexa- and octa-

decenoic FA positional isomers.

Keywords Euonymus species � Neutral acylglycerols �1,2,3-Triacyl-sn-glycerols � 3-Acetyl-1,2-diacyl-sn-

glycerols � Fatty acids � Taxonomic position

Abbreviations

amu Atomic mass unit

DW Dry weight

FA Fatty acid(s)

Pam Palmitic, 16:0

Pol D9-Hexadecenoic, D9-16:1

Ste Stearic, 18:0

Ole Oleic, D9-18:1

Vac cis-Vaccenic, D11-18:1

Lin Linoleic, D9,12-18:2

Lnn a-linolenic, D9,12,15-18:3

FAME FA methyl ester(s)

acDAG 3-acetyl-1,2-diacyl-sn-glycerol(s)

DMOX 4,4-Dimethyl-2-oxazoline

NAG Neutral acylglycerol(s)

TAG 1,2,3-Triacyl-sn-glycerol(s)

UI Unsaturation index

Introduction

The genus Euonymus L. has the largest number of species

in the Celastraceae family. It consists of shrubs or woody

plants and is distributed mostly in the Northern Hemi-

sphere including Russia [1]. To judge from various sources,

it includes 129 [2], 176 [1], or even 220 species [3].

According to a most recent taxonomy system [2], these

species are subdivided into two subgenera, Euonymus and

Kalonymus, and a number of sections. Most Euonymus

species occur in Asia (115 species, *88.5 %), while

Europe and Central America include four species each [2];

more than 40 species are cultivated in Europe and North

America as ornamental plants and green fences [4].

R. A. Sidorov � A. V. Zhukov � V. P. Pchelkin �A. G. Vereshchagin � V. D. Tsydendambaev (&)

Laboratory of Lipid Metabolism, K. A. Timiryazev Institute of

Plant Physiology, Russian Academy of Sciences,

Botanicheskaya 35, Moscow 127276, Russia

e-mail: vdt@ippras.ru; vdt1952@mail.ru

123

J Am Oil Chem Soc

DOI 10.1007/s11746-014-2425-2

Up till now, phytochemical investigations of many

species of the genus Euonymus differing from each other in

the morphology of their fruits were performed mostly for

detecting bioactive substances. All parts of these plants are

used in Chinese folk medicine, because of their chemical

constituents exhibiting antitumor, antimicrobial, antidia-

betes, and insecticidal effects [3]. The phytochemical

studies resulted in the isolation and identification of more

than 200 chemical constituents, including 43 triterpenoids,

82 sesquiterpenes, 22 flavonoids, 11 fatty acids (FA), 40

alkaloids, six steroids, three cardenolides, five lignanoids,

and 34 other compounds [3].

Meanwhile, it has long been known that the fruits of

these plants contain fatty oil [1]. The oil has been shown to

be accumulated not only in the seeds, but also in the fruit

arils. Oil-containing oleosomes have been detected in the

aril epidermal cells of E. europaeus [5], and such oleo-

somes are also present in the aril parenchyma cells of

several Euonymus species [6]. Thus, the plants of the genus

Euonymus L. belong to the group of plants with an oil-

bearing mesocarp [7]. These plants accumulate oil not only

in the seeds, which desiccate during maturation, but also in

the water-saturated fruit parts other than seeds and pos-

sessing only a maternal genotype, such as mesocarps,

hypanthia, and arils. Moreover, it turned out that these

parts of Euonymus fruit sharply differ from each other in

the composition of the fatty oil.

In the seeds of Euonymus species studied earlier, the FA

neutral acylglycerols (NAG) of the oil contained no more

than 2–15 % of common 1,2,3-triacyl-sn-glycerols (TAG)

of usual composition typical for nearly all oil-bearing

plants, but up to 80–98 % of unusual, optically active

3-acetyl-1,2-diacyl-sn-glycerols (acDAG). At the same

time, aril NAG consisted mainly of the usual TAG, which

were accompanied by only a small amount of acDAG [8,

9].

Up till now, the FA composition of NAG classes (TAG

and acDAG) of seeds and arils was studied to some extent

only in a few Euonymus species [10]. Meanwhile, many

representatives of this genus are known (see above) to

differ greatly from each other in the morphology of fruits

and seeds [1, 2, 4, 5]. Therefore, it was of interest to

investigate the special features of NAG composition in the

fruits of a number of other Euonymus species [1]. Under

conditions of modern normal-phase preparative and ana-

lytical TLC, TAG markedly exceeded acDAG in the extent

of their mobility [8]. Therefore, these convenient and

reliable techniques were successfully used in our studies

for the isolation and purification of NAG.

This paper is devoted to determining the dry matter

content, absolute and relative content of NAG classes and

their proportions as well as total FA compositions in

mature seeds and arils. The 14 Euonymus species studied

here showed themselves to differ considerably in the above

parameters.

Materials and Methods

Plant Material

Mature fruits of 14 species belonging to four sections and

two subgenera of the genus Euonymus (Table 1) were

collected during 2010–2011 in the arboretum of the Main

Botanical Garden of the Russian Academy of Sciences

[10]. Herbarium voucher specimens were deposited in the

herbarium of this garden (Moscow). Every fruit sample

was taken from 3 to 4 plants of a given species.

N seeds ? arils (as a whole) were obtained from fruits, and

the arils (flesh) were separated. The means of fresh (mFW,

mg) and dry matter (mDW = mFW 9 WD/100) mass of a

single seed and a single aril (WD, %—content of dry

matter in the total fresh seeds ? arils) were calculated.

Fresh seeds ? arils (N = 100) stored at -20 �C were

quickly weighed and immediately fixed for 1 min with

boiling water to inactivate the enzymes. Separated seeds

(mFW, g) were washed with purified chloroform and, if

necessary, stored at -20 �C.

Extraction of Lipids and Isolation of NAG

Fresh flesh (mFW, g) mixed with washing liquid was

homogenized with water. Total lipids were extracted by

chloroform containing 0.001 % BHT from the aqueous

homogenate in a separatory funnel. The lipid residue was

weighed, dissolved in V ml of benzene and stored as a

benzene solution at -20 �C. Fresh seeds were treated in a

same way.

After removing benzene in vacuo, the lipids were dis-

solved in n-hexane and transferred onto a neutral alumina

column [10]. The column was washed with n-hexane-

benzene mixtures, first 12:1 (v/v, 100 ml) and then 6:4 (v/

v, 150 ml), and all eluates containing NAG were collected.

In order to isolate the preparations of separate NAG classes

(TAG and acDAG), the aliquots of n-hexane-benzene

eluates (v ml, *75 mg of lipids) were fractionated by

preparative TLC using a 20 9 20-cm Kieselgel 60F

(Merck) plate preimpregnated with a 0.001 % 20,70-dichlorofluorescein (Sigma) solution in ethanol, the n-

hexane–diethyl ether system (95:5 v/v) serving as a mobile

phase. The NAG zones were viewed under UV light

(k = 254 nm), transferred onto glass filters, and eluted

with a chloroform–methanol mixture, 96:4 v/v containing

0.001 % BHT. The degrees of purity of NAG preparations

thus obtained were checked by analytical TLC using

4 9 10 cm Silufol plates [10].

J Am Oil Chem Soc

123

Determination of Content and FA Composition of NAG

The weight of esterified FA in each NAG class (W, lg) was

estimated by quantitative GC–MS analysis of FA methyl

esters (FAME) using an internal standard technique [10].

An absolute quantity of these classes in a single seed and/or

aril (P, lg/total number of seeds or arils) was calculated by

the equation

P ¼ V �Wð Þ= v� Nð Þ:

Qualitative and quantitative FA composition in TAG

preparations and in sn-1,2-positions of acDAG was deter-

mined by GC–MS [10], For determining the structure of two

unusual FA designated by us earlier as X1 and X2, they were

converted into their 4,4-dimethyl-2-oxazoline (DMOX)

derivatives [11], which were subsequently analyzed by GC–

MS using an Agilent 7890A GC device (Agilent Technolo-

gies, USA) fitted with a capillary column (DB-23, Ser. no.

US8897617H, 60 m 9 0.25 mm) containing a grafted

Table 1 Content and fatty acid composition of separate classes of NAG (TAG and sn-1,2-positions of acDAG) from the seeds of mature fruits of

14 Euonymus species

Euonymus species NAG classes NAG(mg/g DW) Fatty acids (mass. %) UI

Pam Pol Ste Ole Vac Lin Lnn Othersa

Section Euonymus

E. bungeanus TAG 12.6 23.0 1.4 4.1 31.5 1.4 32.6 2.8 3.2b 1.097

acDAG 116.4 15.6 0.2 1.8 38.4 1.2 34.6 7.6 0.6 1.320

E. europaeus TAG 1.6 22.4 2.3 4.0 31.9 2.8 25.9 4.4 6.3c 0.912

acDAG 29.7 18.1 0.2 2.0 43.8 1.1 24.6 9.3 0.9d 0.951

E. hamiltonianus TAG 3.1 22.5 0.7 3.2 33.0 1.1 33.4 4.3 1.8 1.153

acDAG 41.9 16.3 0.1 2.7 52.5 1.1 19.6 7.0 0.7 1.141

E. phellomanus TAG 3.4 23.6 0.4 4.9 31.9 0.9 32.0 4.9 1.4 1.121

acDAG 46.9 19.1 0.1 2.3 47.4 1.2 21.2 8.1 0.6 1.154

E. semiexsertus TAG 11.9 22.4 1.5 2.8 29.9 1.9 34.9 4.6 2.0 1.181

acDAG 133.5 16.4 0.1 2.2 50.6 1.0 20.5 8.7 0.5 1.189

E. sieboldianus TAG 10.9 20.8 8.0 2.4 23.7 7.2 32.3 2.4 3.2 1.135

acDAG 128.4 15.4 0.1 2.0 54.8 1.2 19.2 6.8 0.5 1.152

Section Melanocarya

E. alatus TAG 5.9 24.8 0.2 6.3 31.0 1.0 32.9 1.8 2.0 1.035

acDAG 152.0 20.2 0.1 2.9 25.7 0.9 47.9 1.7 0.6 1.279

E. sacrosanctus TAG 6.6 18.6 0.3 4.9 36.8 1.1 35.5 1.8 1.0 1.147

acDAG 200.8 18.7 0.2 2.3 27.0 1.0 48.8 1.7 0.3 1.309

Section Pseudovyenomus

E. pauciflorus TAG 8.8 15.6 0.3 5.3 36.5 0.8 36.9 2.3 2.3 1.186

acDAG 239.8 15.5 0.1 3.5 29.4 0.7 48.7 1.4 0.7 1.320

E. verrucosus TAG 4.7 16.7 0.2 4.8 48.6 0.8 25.1 1.8 2.0 1.057

acDAG 154.4 17.3 0.1 3.6 44.0 0.7 31.0 2.0 1.3 1.133

Section Kalonymus

E. latifolius TAG 6.4 16.6 0.2 5.8 52.0 1.1 16.3 6.0 2.0 1.044

acDAG 141.0 17.7 0.1 2.6 46.1 1.2 25.4 6.0 0.9 1.167

E. macropterus TAG 2.4 22.2 0.5 3.9 42.2 1.4 25.3 2.9 1.6 1.039

acDAG 51.2 18.6 0.2 2.5 43.8 1.1 26.1 7.2 0.6 1.191

E. maximoviczianu s TAG 2.9 21.9 0.3 5.3 43.9 1.2 22.9 3.1 1.4 1.008

acDAG 33.7 19.4 0.2 4.3 36.8 1.0 31.2 6.9 0.2 1.199

E. sachalinensis TAG 0.2 28.0 0.3 4.4 44.1 1.1 14.8 5.3 2.0 0.917

acDAG 31.9 20.9 0.0 3.2 54.1 1.1 14.1 6.0 0.6 1.014

a Sum of other FA (10:0, 12:0, 14:0, 15:0, D7-16:1, D7,10-16:2, D9,12-16:2, 17:0, 20:0, D11-20:1, 22:0); the concentration of each of them was

usually \1.0 %b,c,d D8-16:1 acid was present (see Table 4)c The concentration of D11-16:1 acid was 2.3 %

J Am Oil Chem Soc

123

(50 % cyanopropyl)-methylpolysiloxane polar liquid phase

as a 0.25 mm-thick film. The FAME were separated under

the following conditions: carrier gas (helium) pressure in the

injector, 191 kPa; operational gas pressure in the column at

1 mL/min, 245 kPa; carrier gas flow linear velocity in the

column, 18 cm/s; sample volume, 1 lL (10 lg FAME); flow

split ratio, 1:20; evaporator temperature, 260 �C. The oven

temperature program was as follows: from 130 to 170 �C at

6.5 �C/min, to 215 �C at 2.75 �C/min (25 min at this tem-

perature), to 240 �C at 40 �C/min, and 50 min at 240 �C,

operational temperature of the mass selective detector

(5975C MSD), 240 �C. For identifying individual FAME

species and calculating their concentrations in the mixture, a

NIST search library and from the MSD Chem Station

G1701EA E.02.00.493 were used [10].

The unsaturation index (UI) of FA mixtures was cal-

culated as follows: UI = R pi 9 ei/100, where pi is the

percentage and ei—the double bond number of i-th FA.

Statistical Analysis

All experiments were performed with three replicates.

Tables and figures show the means of P weight values; in

all cases, the SD did not exceed 7 % of a mean value.

Normality of distribution of experimental values in sam-

ples was tested using a Shapiro–Wilk criterion. All

numerical values obtained here—dry matter content, %;

concentrations of i-th FA, mass. %; and unsaturation index,

relative units—were characterized by normal distribution

(0.79 C W [ 1.0, 0.26 [ p [ 0.56). Significant differ-

ences between means were tested by dispersion analysis

(one-way analysis of variance, ANOVA). The extent of

possible relationships between the Euonymus species

studied here is based on similarities in FA compositions of

aril TAG and was established by hierarchical cluster ana-

lysis—an unweighted pair-group method with arithmetic

averaging (UPGMA), and also by clustering using a k-

means concept. All statistical analyses were carried out

with the software Statistica v. 10 programs (StatSoft,

USA).

Results and Discussion

Content of Dry Matter and NAG in Fruits

The dry matter contents of seeds and arils of mature fruits

of 14 Euonymus species are shown in Fig. 1. From these

data, it could be calculated that, in the absolute and relative

dry matter content, the seeds exceeded the arils on an

average 2.9- and 1.9-fold, respectively. Typically, Euony-

mus species differed considerably in the absolute dry

matter content (mg/fruit part) and in the relative dry matter

content in the arils; however, most were similar in dry

matter content in seeds.

The data on TAG and acDAG content in Euonymus

fruits are presented in Fig. 2 and Tables 1 and 2. Using

these data, it could be calculated that, on the average, the

level of NAG in the arils, as a percentage of dry matter,

was almost twice as high as in the seeds (21.9 and 11.3 %

seeds and arils, respectively). However, if calculated as mg

per given fruit part the contents were 2.6 and 3.3 mg, seeds

Fig. 1 Absolute (a) and relative (b) dry matter contents in the fruit seeds and arils of 14 Euonymus species

J Am Oil Chem Soc

123

and arils, respectively. This discrepancy was brought about

by differences between seeds and arils in the dry matter

content (see above). When using both of these calculation

techniques, the proportion of TAG in seeds comprised

4–5 %, and in arils, *98 % of total NAG. As shown by

Durrett et al. [9], the concentrations of TAG and acDAG in

the seeds of E. alatus were 3.3 and 91.7 %, and in the arils,

91.2 and 0.5 % of total lipids, respectively. It is seen that,

in Euonymus fruits, a conventional qualitative composition

of NAG, namely a predominance of TAG, is observed only

in the arils, which possess only a maternal genotype (see

above), while in the seeds, which include the genes of both

parents, are present almost exclusively acDAG rather

rarely occurring in plants.

Previously, we had shown that the seeds and water-

saturated hypanthia of sea buckthorn fruits, whose NAG

were comprised only of TAG, also sharply differed in the

qualitative FA composition of these lipids. However, in

that case, a conventional qualitative FA composition

comprising mainly oleate, linoleate, and linolenate was

found in the seeds, while the TAG of hypanthia were

characterized by a predominance of palmitate as well as

unusual monounsaturated FA, namely D9-hexadecenoic

and cis-vaccenic [12].

In all Euonymus species studied here (Tables 1, 2;

Fig. 2), the TAG, within certain amounts, occurred in both

parts of the fruit. Meanwhile, the acDAG were always

found only in seeds, while in the arils of four species, they

were almost or totally absent (Table 2). These differences

were likely to be caused by a reduced activity of acDAG

biosynthesis in the arils (see above). It must be stressed that

there are also other differences between individual Euon-

ymus species in their ability to form separate NAG classes,

namely a greatly reduced TAG level in E. sachalinensis

seeds (Fig. 2b) and E. verrucosus arils (Fig. 2a), as well as

an unusually high absolute content of acDAG in E.

latifolius seeds (Fig. 2b).

FA Composition of NAG in the Fruits of the 14

Euonymus Species

In the NAG of fruits, 14 molecular species of FA

(excluding minor ones) were found, with a predominance

of linoleic, oleic, palmitic, and a-linolenic acids (Tables 1,

2). More or less noticeable amounts (up to 12 %) of unu-

sual D9-hexadecenoic, D9,12-hexadecadienoic (palmitoli-

noleic), and D11-octadecenoic (cis-vaccenic) acids, as well

as previously unidentified X1 and X2 acids were also

present. As shown in Figs. 3, 4a, b, X1 and X2 acids were

identified as D8-hexadecenoic and D10-octadecenoic acids,

respectively. This statement was conclusively substantiated

by the fact that the diagnostic ions for D8-16:1 acid in

Fig. 4a—168, 182, 194, 208, 232,�, 307 (M?)—differed

from the corresponding ions for D10-18:1 acid in Fig. 4b—

196, 210, 222, 236, 250, …, 335 (M?)—by 28 amu The

MS of DMOX-X2 was identical to that of DMOX deriva-

tive of D10-18:1 acid displayed in the Lipid Library [11],

while that of DMOX-D8-16:1 acid was absent in [11].

Previously, it had been shown that, in the sn-1,2-posi-

tions of acDAG from E. verrucosus seeds, the content of

palmitate, stearate, oleate, linoleate, and linolenate was 14,

4, 40, 40 and 2 % [8], while for three species of the genus

Maytenus also belonging to Celastraceae, the respective

values were 13.0–15.2, 3.0–6.0, 36.8–58.7, 21.0–44.5, and

0.2–0.6 % [13]. This evidence indicates that there is a

certain similarity between different genera in the family

Fig. 2 Absolute neutral acylglycerol (NAG) contents in the arils (a) and seeds (b) of 14 Euonymus species

J Am Oil Chem Soc

123

Celastraceae regarding the major FA composition of NAG

in their fruits.

In all NAG samples (Tables 1, 2), except those from E.

hamiltonianus seeds (Table 1), the TAG were, to a certain

extent, below the acDAG in their UI value. Such differ-

ences seem to be caused, first of all, by the fact that a TAG

molecule contains two positions of glycerol residue (sn-1

and sn-3) specific to less unsaturated FA [12], while an

acDAG molecule has only one such position (sn-1) [8, 9].

The difference found here was expressed much more

intensively in arils, where mean UI values in TAG and

acDAG were 1.051 and 1.357, respectively, whereas the

Table 2 Content and fatty acid composition of separate classes of NAG (TAG b sn-1,2-positions of acDAG) from the arils of mature fruits of 14

Euonymus species

Euonymus species NAG classes NAG (mg/g DW) Fatty acids (mass. %) UI

Pam Pol Ste Ole Vac Lin Lnn Othersa

Section Euonymus

E. bungeanus TAG 163.0 29.3 3.9 1.4 11.2 3.0 39.7 1.2 10.3b 1.109

acDAG 3.5 20.5 1.5 1.5 10.9 2.2 53.6 2.7 7.1c 1.351

E. europaeus TAG 102.2 23.0 3.8 1.7 17.8 4.6 41.4 2.3 5.4d 1.147

acDAG 3.4 21.3 2.6 1.6 10.0 4.0 52.7 2.7 5.1e 1.340

E. hamiltonianus TAG 276.0 30.8 1.8 2.2 15.3 1.7 45.0 1.6 1.6 1.142

acDAG 6.4 19.9 0.7 1.8 7.4 1.5 64.9 2.4 1.4 1.468

E. phellomanus TAG 115.4 32.9 1.2 4.7 16.6 1.0 39.5 1.7 2.4 1.037

acDAG – – – – – – – – – –

E. semiexsertus TAG 437.6 29.4 3.8 1.4 11.4 4.0 46.5 1.1 2.4f 1.179

acDAG 12.1 20.7 1.3 1.6 5.7 3.5 63.2 1.8 2.2g 1.451

E. sieboldianus TAG 222.8 21.7 12.6 1.7 13.2 10.7 34.5 1.0 4.6h 1.137

acDAG 5.0 15.9 7.1 1.2 4.5 8.8 53.5 2.3 6.7i 1.425

Section Melanocarya

E. alatus TAG 196.1 26.3 0.4 1.8 21.4 1.5 45.2 2.7 0.7 1.219

acDAG 1.2 23.0 0.3 2.4 19.8 1.6 47.0 3.3 2.6 1.261

E. sacrosanctus TAG 302.8 23.5 0.9 1.8 25.3 2.1 43.1 2.8 0.5 1.231

acDAG 3.3 17.6 0.6 2.3 15.2 1.7 43.8 14.3 4.5 1.495

Section Pseudovyenomus

E. pauciflorus TAG 305.7 27.9 0.9 4.9 36.8 1.4 25.6 1.0 1.5 0.933

acDAG 1.6 18.4 0.4 3.4 30.4 0.9 40.0 2.5 4.0 1.202

E. verrucosus TAG 0.3 11.0 2.6 2.6 38.5 3.5 26.0 3.8 12.0k 1.095

acDAG – – – – – – – – – –

Section Kalonymus

E. latifolius TAG 199.7 21.6 1.4 1.9 52.3 1.0 18.1 2.7 1.0 0.994

acDAG 2.6 17.2 0.7 2.9 42.2 0.9 25.6 8.0 2.5 1.197

E. macropterus TAG 324.2 41.8 0.4 2.8 18.9 1.7 31.1 2.7 0.6 0.915

acDAG 1.8 22.5 0.7 3.1 19.2 1.4 32.3 17.3 3.5 1.384

E. maximoviczianu s TAG 191.7 39.0 0.8 3.2 27.5 0.7 26.9 1.0 0.9 0.862

acDAG – – – – – – – – – –

E. sachalinensis TAG 181.0 35.6 0.4 5.5 44.5 1.5 11.4 0.7 0.4 0.713

acDAG – – – – – – – – – –

‘‘–‘‘ implies not founda Sum of other FA (10:0, 12:0, 14:0, 15:0, D11-16:1, D7,10-16:2, 17:0, 20:0, D11-20:1, 22:0, 24:0); the concentration of each of them was

usually \1.0 %b,c,d,e,f,g,h,i D8-16:1 acid was present (see Table 4)b,c D10-18:1 acid was present (see Table 4)d,e Concentrations of D7-16:1 acid were 3.9 and 2.6 %, respectivelyg,I Concentrations of D9,12-16:2 acid were 1.2 and 2.9 %, respectivelyk Concentrations of 12:0 and 14:0 acids were 4.8 and 3.1 %, respectively

J Am Oil Chem Soc

123

values in seeds were 1.074 and 1.180, respectively; in this

case, it was brought about by a much higher level of

linoleate in the acDAG of arils as compared to the acDAG

of seeds (see Tables 1, 2). In both fruit parts, TAG usually

exceeded acDAG in the content of saturated FA and ranked

below them as regarding the level of unsaturated FA

(Tables 1, 2).

Characteristics of Major Fatty Acid Composition

of NAG from Separate Subgenera and Sections

of the Genus Euonymus

When considering the data of Tables 1 and 2, it was

established that, in several cases, there was a definite

relationship between the major FA composition of NAG

in certain plant species and their taxonomic position in the

genus Euonymus. Separate sections of the genus Euony-

mus could differ somewhat in the content of individual

molecular species of unsaturated FA in total seed and aril

NAG.

As shown in Table 2, TAG greatly predominated in the

aril NAG. In order to reveal possible relationships between

the FA composition of these TAG and the taxonomic

position of separate Euonymus species (Table 2), we per-

formed a hierarchical cluster analysis (Fig. 5); E. ver-

rucosus was excluded from this analysis, because the arils

of its fruits were virtually devoid of oil (0.02–0.10 % of

DW). From Fig. 5, it is seen that the remainder of the 13

Euonymus species were grouped into three clusters.

All species in cluster 1 belonged to the subgenus

Euonymus, while those in the clusters 2 and 3, except E.

pauciflorus, belonged to the subgenus Kalonymus (see

Table 1 in [14]). According to current classification [1], E.

pauciflorus has been assigned to the subgenus Euonymus,

but, in compliance with modern evidence, it is more similar

to representatives of the subgenus Kalonymus not only in

its aril TAG composition, but also in the anatomical

structure of its mature arils [15].

In order to determine the factors, which brought about

the formation of clusters 1 and 2 and 3, we performed an

additional analysis of FA composition of aril TAG using k-

means concept (Fig. 6). It was definitely demonstrated that

the clusters were grouped according to such significant

factors as average concentrations of oleic (p = 0.0001,

F = 32.908) and linoleic acids (p = 0.0001, F = 33.801)

in aril TAG. The species in the subgenus Euonymus

(cluster 1) were characterized by an increased content of

linoleate, while those from Kalonymus (clusters 2 and 3),

by the predominance of oleate. Such conclusion was

additionally confirmed by dispersion analysis of unsatura-

tion index values of aril TAG: in this regard, the subgenus

Euonymus species significantly (p = 0.001, F = 17.866)

exceeded those from subgenus Kalonymus. (UI =

1.05–1.20 and 0.75–1.00, respectively).

Species belonging to cluster 1 also differed from those

of clusters 2 and 3 by a somewhat enhanced content of D9-

hexadecenoic and cis-vaccenic acids (Fig. 6), but these

differences were not significant. No differences regarding

palmitate and stearate content were observed.

Corresponding cluster analysis of FA composition of

acDAG from seeds (Table 1) did not reveal any taxo-

nomical relationships, while no analyses of seed TAG and

aril acDAG were carried out because of their low con-

centrations in these fruit parts (Tables 1, 2).

Fig. 3 GC separation of the FAME obtained from the aril TAG of E. bungeanus. IS internal standard

J Am Oil Chem Soc

123

Special Features of NAG Unusual Fatty Acid

Composition in the Section Euonymus of the Genus

Euonymus

Certain relationship between a taxonomic position of plants

and the FA composition of their fruit NAG was established

not only for major FA, but also for some unusual FA

species present in NAG in rather moderate amounts. These

FA were comprised of a number of positional isomers of

hexa- and octadecenoic FA, namely D9-hexadecenoic and

cis-vaccenic acids (Tables 1, 2, 3) as well as D8-hexa-

decenoic and D10-octadecenoic acids (Table 4), the section

Euonymus sharply differing from the rest of sections

studied here in their increased concentration in NAG.

Within this section, E. hamiltonianus and E. phellom-

anus ranked below four other species in the FA positional

Fig. 4 The mass spectra of 4,4-

dimethyl-2-oxazoline (DMOX)

derivatives of a D8-

hexadecenoate (X1-FA) and

b D10-octadecenoate (X2-FA)

Fig. 5 Dendrogram of the 13 Euonymus species based on the linkage

distance and resulting from the cluster analysis of these species as

regards the FA composition of aril TAG (unweighted pair-group

method with arithmetic averaging, Chebyshev distance metric)

J Am Oil Chem Soc

123

isomer content, being totally devoid of D8-hexa- and D10-

octadecenoic acids. Arils and TAG typically exceeded

seeds and acDAG, respectively, in the concentration of

positional isomers, while cis-vaccenic acid usually sur-

passed D9-hexadecenoic acid in its quantitative content. An

enhanced level of these two FA was particularly typical for

the NAG from E. sieboldianus.

As stated above, D8-hexa- and D10-octadecenoic acids

occurred only in four Euonymus species and mostly in

TAG (Table 4), their content being far less than that of D9-

hexadecenoic and cis-vaccenic acids. The D10-18:1 acid

was found only in E. bungeanus aril NAG, and the content

of D8-16:1 acid in the arils was higher than in the seeds.

In plants, D9-hexadecenoic and cis-vaccenic acids are

known to be related to each other in the mechanism of their

biosynthesis, because cis-vaccenic acid can be formed only

via C2-elongation of D9-hexadecenoic [16]. It can be

suggested that, by analogy with them, D10-octadecenoic

acid might be formed by C2-elongation of D8-hexadece-

noic acid. So far as we know, these FA had never been

found previously in higher plants. At the same time, D9-

hexadecenoic and cis-vaccenic acids were repeatedly

demonstrated in the fruits of other plants with an oil-

bearing mesocarp, including the sea buckthorn fruits [12].

However, as distinct from Euonymus (see Tables 1, 2),

these acids were localized in sea buckthorn hypanthia and

almost totally absent in the TAG of its seeds [17].

Along with cis-vaccenic acid, a product of elongation of

D9-hexadecenoic acid, the aril acDAG from E. semiex-

sertus and E. sieboldianus, also belonging to the section

Euonymus, containing 1.2–2.9 % of D9,12-hexadecadie-

noic (palmitolinoleic) acid (Table 2) known to be a product

of D12-desaturation of D9-hexadecenoic acid [18]. It

should also be noted (Tables 1, 2) that E. europaeus, still

another representative of the section Euonymus, contained

two additional 16:1 positional isomers, namely, D7-hexa-

decenoic acid in the TAG and acDAG of their arils (3.9

and 2.9 %, respectively) and D11-hexadecenoic acid in the

TAG of their seeds (2.3 %).

Recently, we established [10, 14] that the seeds and arils

of several Euonymus species are characterized by the pre-

sence of FA lower alkyl esters and, in particular, FA

methyl esters (FAME). These esters, shown to represent the

products of natural biosynthesis rather than experimental

artifacts, are very rarely found in higher plants. Later, we

demonstrated that E. sieboldianus and E. phellomanus

(section Euonymus) differed from almost all other Euony-

mus species studied in our work in the qualitative FAME

Fig. 6 k-Means values of i-th FA content for the clusters 1 and 2 and

3 (Fig. 5). FA designations: 1—10:0, 2—12:0, 3—14:0, 4—16:0, 5—

D7-16:1, 6—D8-16:1, 7—D9-16:1, 8—D7,10-16:2, 9—D9,12-16:2,

10—18:0, 11—D9-18:1, 12—D10-18:1, 13—D11-18:1, 14—D9,12-

18:2, 15—D9,12,15-18:3, 16—20:0, 17—D11-20:1, 18—D11,14-

20:2, 19—22:0

Table 3 Average concentrations of D9-hexadecenoic (D9-16:1) and

cis-vaccenic (D11-18:1) acids in total TAG and total acDAG from the

seeds and arils of mature fruits of species belonging to various sec-

tions of the genus Euonymus

Plant species Classes

of NAG

Seedsa Arilsb

Fatty acids (mass. %)

D9-

16:1

D11-

18:1

D9-

16:1

D11-

18:1

Total species of the section

Euonymus

TAG 2.1 2.6 4.5 4.2

acDAG 0.1 1.1 2.6 4.0

Total species of the sections

Melanocarya,

Pseudovyenomus and

Kalonymus

TAG 0.3 1.1 1.0 1.7

acDAG 0.1 0.9 0.5 1.5

a Calculated from Table 1b Calculated from Table 2

Table 4 Concentration (mass. %) of D8-16:1 and D10-18:1 acids in

TAG and acDAG from the seeds and arils of separate species of the

section Euonymus

Euonymus species NAG classes Seedsa Arilsb

D8-16:1 D8-16: D10-18:1

E. bungeanus TAG 1.1 6.7 2.2

acDAG – 3.8 1.4

E. europaeus TAG 0.7 3.8 –

acDAG 0.2 – –

E. semiexsertus TAG – 0.7 –

acDAG – 0.6 –

E. sieboldianus TAG – 2.3 –

acDAG – 1.6 –

a Calculated from Table 1b Calculated from Table 2

J Am Oil Chem Soc

123

composition in the arils of their fruits. These FAME con-

tained the esters of D9-hexadecenoic and cis-vaccenic

acids, and concentrations of these esters in E. sieboldianus

were 0 and 1.6 %, and in E. phellomanus, 3.3 and 2.8 %,

respectively [14].

One can see that the fruits of the section Euonymus and,

particularly, their arils are characterized by an enhanced

capability of forming a number of hexa- and octadecenoic

FA positional isomers. Among them, D8- and D9-hexa-

decenoic acids were further subjected to C2-elongation, and

D9-hexadecenoic acid, to D12-desaturation. The unusual

FA could incorporate not only in the NAG, but also in the

FAME naturally occurring in both fruit parts [10].

Conclusion

At present, acDAG show promise as a raw material for

biodiesel fuel, because of their low viscosities, i.e. they

rank 30 % below those of the usual TAG [9]. Another

possible acDAG usage is as a substitute for common fats in

the human diet since their calorie content is much lower

than that of TAG of usual composition (5 and 9 kcal/g,

respectively) [9, 19]. Within the last few years, acDAG of

synthetic origin have also been investigated. These prepa-

rations are known as SALATRIM�, a commercial product,

in which two OH groups of a glycerol residue are esterified

with higher FA and one OH group, with acetic, propionic

or butyric acid [19, 20]. It was shown that the SALA-

TRIM� meal increased fullness (P = 0.04) and decreased

hunger (P = 0.06) significantly more than did the tradi-

tional fat meal [20]. AcDAG of Celastraceae plants are the

natural analogs of the SALATRIM� product, and therefore

investigations of their quantitative content and FA com-

position in various fruit parts may be of practical

importance.

The structural analyses of molecular species of TAG and

acDAG from these plants will be the subject of our future

work.

Acknowledgments We are grateful to Dr. N.A. Trusov, Main

Botanical Garden of RAS, for his help in obtaining plant material.

References

1. Blakelock RA (1951) A synopsis of the genus Euonymus L. Kew

Bull 2:210–290

2. Ma JS (2011) A revision of Euonymus (Celastraceae). Thaizia

11:1–264

3. Zhu J-X, Qin J-J, Chang R-J, Zeng Q, Cheng X-R, Zhang F, Jin

JH-Z, Zhang W-D (2012) Chemical constituents of plants from

the genus Euonymus. Chem Biodivers 9:1055–1076. doi:10.1002/

cbdv.201100170

4. Simmons MP, McKenna MJ, Bacon CD, Yakobson K, Cappa JJ,

Archer RH, Ford AJ (2012) Phylogeny of Celastraceae tribe

Euonymeae inferred from morphological characters and nuclear

and plastid genes. Mol Phylogenet Evol 62:9–20. doi:10.1016/j.

ympev.2011.08.022

5. Corner EJH (1976) The seed of dicotyledons, vol 1. Cambridge

Univ. Press, London

6. Melikyan AP, Savinov IA (2000) Family Celastraceae. In: Tak-

htajan AL (ed) Comparative anatomy of seeds. Nauka, St.

Petersburg, pp 123–135 (in Russian)

7. Berezhnaya GA, Yeliseev IP, Ozerinina OV, Tsydendambaev

VD, Vereshchagin AG (1993) Developmental changes in the

absolute content and fatty acid composition of acyl lipids in sea

buckthorn fruits. Plant Physiol Biochem 31:323–332

8. Kleiman R, Miller RW, Earle FR, Wolff IA (1967) (S)-1,2-

Diacyl-3-acetins: optically active triglycerides from Euonymus

verrucosus seed oil. Lipids 2:473–478. doi:10.1007/BF02533174

9. Durrett TP, McCloskey DD, Tumaney AW, Elzinga DA, Ohl-

rogge J, Pollard M (2010) A distinct DGAT with sn-3 acetyl-

transferase activity that synthesizes unusual, reduced-viscosity

oils in Euonymus and transgenic seeds. Proc Nat Acad Sci USA

107:9464–9469. doi:10.1073/pnas.1001707107

10. Sidorov RA, Zhukov AV, Vereshchagin AG, Tsydendambaev

VD (2012) Occurrence of fatty acid lower alkyl esters in Euon-

ymus fruits. Russ J Plant Physiol 59:326–332. doi:10.1134/

S1021443712030156

11. Christie WW (2012), www.lipidlibrary.aocs.org/ms/masspec.

html

12. Pchelkin VP, Kuznetsova EI, Tsydendambaev VD, Vereshchagin

AG (2006) Distribution of unusual fatty acids in the triacylgly-

cerols of sea buckthorn mesocarp oil. Russ J Plant Physiol

53:346–354. doi:10.1134/S1021443710060142

13. Spitzer V, Aichholz R (1996) Analysis of naturally occurring a-

acetotriacylglycerides by gas chromatography—chemical ioni-

zation mass spectrometry. J High Resol Chromatogr 19:497–502.

doi:10.1002/jhrc.1240190905

14. Sidorov RA, Zhukov AV, Pchelkin VP, Vereshchagin AG, Tsy-

dendambaev VD (2013) Occurrence of fatty acid short-chain-

alkyl esters in fruits of Celastraceae plants. Chem Biodivers

10:976–988. doi:10.1002/cbdv.201200329

15. Trusov NA (2010) Morphological and anatomical structure of

fruits of the representatives of the family Celastraceae R. Br. in

connection with their oil content, PhD Thesis. Main Botanical

Garden, Russian Academy of Sciences, Moscow (in Russian)

16. Shibahara A, Yamamoto K, Takeoka M, Kinoshita A, Kajimoto

G, Nakayama T, Noda M (1989) Application of a GC–MS

method using deuterated fatty acids for tracing cis-vaccenic acid

biosynthesis in kaki pulp. Lipids 24:488–493. doi:10.1007/

BF02535127

17. Mallet G, Dimitriades C, Ucciani E (1988) Quelques examples de

repartition entre pulpe et graines des acides palmitoleique et cis-

vaccenique. Rev Franc Corps Gras 35:479–483

18. Shirasaka N, Umehara T, Murakami T, Yoshizumi H, Shimizu S

(1998) Microbial conversion of palmitoleic acid to 9,12-hex-

adecadienoic acid (16:2x4) by Trichoderma sp. J Amer Oil Chem

Soc 75:717–720. doi:10.1007/s11746-998-0211-8

19. Smith RE, Finley JW, Leveille GA (1994) Overview of SALA-

TRIM, a family of low-calorie fats. J Agric Food Chem

42:432–434. doi:10.1021/jf00038a036

20. Sørensen LB, Cueto HT, Andersen MT, Bitz C, Holst JJ, Rehfeld

JF, Astrup A (2008) The effect of Salatrim, a low-calorie modi-

fied triacylglycerol, on appetite and energy intake. Am J Clin

Nutr 87:1163–1169

J Am Oil Chem Soc

123