occurrence of fatty acid short-chain-alkyl esters in fruits of celastraceae plants

Occurrence of Fatty Acid Short-Chain-Alkyl Esters in Fruits of CelastraceaePlants

by Roman A. Sidorov, Anatoly V. Zhukov, Vasily P. Pchelkin, Andrei G. Vereshchagin,and Vladimir D. Tsydendambaev*

Laboratory of Lipid Metabolism, Institute of Plant Physiology, Russian Academy of Sciences,Botanicheskaya 35, Moscow 127276, Russia (phone: þ7-499-9778355; fax: þ7-499-9778018;

e-mail: [email protected])

Small amounts of a mixture of fatty acid short-chain-alkyl esters (FASCAEs) were obtained fromthe fruits of twelve plant species of Celastraceae family, and in five of them the FASCAEs were presentnot only in the arils but also in the seeds. These mixtures contained 32 individual FASCAE species, whichformed four separate fractions, viz. FA methyl, ethyl, isopropyl, and butyl esters (FAMEs, FAEEs,FAIPEs, and FABEs, resp.). The FASCAE acyl components included the residues of 16 individual C14 –C24 saturated, mono-, di-, and trienoic FAs. Linoleic, oleic, and palmitic acids, and, in some cases, also a-linolenic acid predominated in FAMEs and FAEEs, while myristic acid was predominant in FAIPEs. Itcan be suggested that, in the fruit arils of some plant species, FAMEs and FAEEs were formed at theexpense of a same FA pool characteristic of a given species and were strongly different from FAIPEs andFABEs esters regarding the mechanism of their biosynthesis. However, as a whole, the qualitative andquantitative composition of various FASCAE fractions, as well as their FA composition, variedconsiderably depending on various factors. Therefore, separate FASCAE fractions seem to besynthesized from different FA pools other than those used for triacylglycerol formation.

Introduction. – Very-long-chain fatty acids (FAs; with the number of C-atoms in analiphatic chain m>18) esterified with higher fatty alcohols (m�20) are known to bethe common components of epicuticular waxes and, therefore, are present, to a certainextent, in most higher plants [1]. Moreover, these esters serve as the major reserve injojoba seeds [2]. At the same time, the esters of common FAs (m�18) with short-chain(m � 4) aliphatic alcohols are rarely present in plant lipids.

Recently [3], by using capillary GC combined with MS, we have shown that neutrallipids of mature and maturing oil-bearing fruits of four euonymus (Euonymus sp.,Celastraceae) species contain small amounts of the mixtures of FA short-chain-alkylesters (FASCAEs). These mixtures are comprised of the esters of common C16 – C18

FAs and four short-chain alcohols, viz. MeOH, EtOH, i-PrOH, and BuOH (i.e.,FAMEs, FAEEs, FAIPEs, and FABEs, resp.). Individual Euonymus species were foundto vary considerably with respect to the qualitative and quantitative composition ofFASCAEs in the arils and seeds of fruits (Fig. 1).

Considering the novelty and variability of our findings, it was of interest to assessthe occurrence of FASCAEs in the fruits of some other Celastraceae species. There-fore, we investigated the composition of FASCAEs in the fruit arils and seeds fromCelastrus rugosus and twelve more Euonymus species belonging to different taxonomicunits [4] . By using experimental approach and evidence published previously

CHEMISTRY & BIODIVERSITY – Vol. 10 (2013)976

� 2013 Verlag Helvetica Chimica Acta AG, Z�rich

[3] [5] [6], it was found that FASCAEs were present in the fruits of most plant speciesstudied here.

The present communication is devoted to the occurrence, identity, and compositionof these FASCAEs.

Results and Discussion. – Occurrence of FASCAEs in Celastraceae Species andIdentification of FASCAEs. Table 1 shows that, of 16 plant species studied in this workand previously [3], only twelve species contained FASCAEs in their fruits. Five ofthem, belonging to the Euonymus and Melanocarya sections of the Euonymussubgenus, contained FASCAEs both in their arils and in the seeds. In these twelvespecies, the yield of FASCAEs from 10 g of fresh arils was ca. 3 – 5 mg, and their contentin the seeds was even lower. A highly purified CHCl3 [7] but no lower alcohols wereused for isolation of FASCAEs from plant material, and a supply of endogenousalcohols from plant material was ruled out (see Exper. Part). Therefore, one can becertain that all these esters, including FAMEs and FAEEs, were natural biosyntheticproducts rather than artifacts of the experiment.

All of four Euonymus species studied earlier contained FASCAEs in the arils, butthey were absent in the seeds of both mature and immature (at the 39th day after theonset of flowering) E. maximowiczianus fruits (see Table 2 in [3]). As shown in Table 1,no FASCAEs were found in the seeds of this species at a later stage of maturation.Thus, E. maximowiczianus seeds were incapable of FASCAE formation regardless ofthe extent of their maturity.

Table 1 also indicates that, in general, FASCAEs were present in the bothsubgenera of the Euonymus genus, as well as in C. rugosus. Only in all threeMelanocarya section species (subgenus Euonymus) and in the two species (out of thesix species studied in this work) of the Euonymus section (from the same subgenus),the FASCAEs occurred in both arils and seeds. At the same time, they were absent inthe species of Pseudovyenomus section, two species of the Euonymus section, and onespecies of the Kalonymus section. Thus, there can be some relationship between thecapability of a plant to form FASCAEs and its taxonomic position.

CHEMISTRY & BIODIVERSITY – Vol. 10 (2013) 977

Fig. 1. a) Transverse and b) longitudinal cross sections of E. maximowiczianus fruit. A, aril; S, seed.


Tabl

e1.

Occ

urre

nce

ofFA

SCA

Es

inA

rils

and

Seed

sof

the

Fru

itsof

16Sp

ecie

sof

Cel

astr

acea

eP

lant

s

Gen

usSu

bgen

usSe

ctio

nSp

ecie

sA

rils

Seed

s

Euo

nym

usE

uony

mus

Euo

nym

usE

.bun

gean

usM

axim

.þ

–E

.eur

opae

usL

.a )þ

þE

.phe

llom

anus

Loes.

þþ

E.h

amilt

onia

nusWall

.þ

–E

.sem

iexs

ertu

sKoehne

––

E.s

iebo

ldia

nusBlume

þ–

Mel

anoc

arya

E.a

latu

s(T

hunb

.)Siebold

þþ

E.s

acro

sanc

tusKoidz

.a )þ

þE

.ala

tus�

E.s

acro

sanc

tusa )

þþ

Pse

udov

yeno

mus

E.p

auci

flor

usM

axim

.–

–E

.ver

ruco

susScop.

––

Kal

onym

usK

alon

ymus

E.l

atif

oliu

sM

ill

.þ

–E

.mac

ropt

erus

Rupr

.þ

–E

.max

imow

iczi

anus

(Prokh

.)Vorosch

.(im

mat

ure)

b)

þ–

E.s

acha

linen

sis

(Fr.Schmidt

)M

axim

.–

–C

elas

trus

Cel

astr

usA

xilla

res

Cel

astr

usru

gosu

sRed

&Wils

þ–

a )D

ata

from

[3].

b)

See

also

[3].

Relative retention times (RRT) of individual FASCAEs identified on the basis of GC/MS evidence are compiled in Table 2. One can see that 32 individual FASCAEs wereidentified in Celastraceae fruits: 16 FAMEs, nine FAEEs, five FAIPEs1), and twoFABEs. The FASCAE acyl components (taking into account also minor ones) includedthe residues of 16 individual C14 – C24 FA species.

Four Euonymus species studied earlier [3] contained only 19 FASCAEs. Theseesters contained, except for traces of myristic acid, the residues of only eight n-C16 – n-C18 FAs.

Composition of Separate FASCAE Fractions and Their FA Composition. Ascompiled in Table 3, all aril FASCAEs contained FAMEs. These FAMEs usuallyexceeded two other fractions in their concentration, and the level of FAEEs in the mostspecies was very moderate (2.6 – 7.8%). At the same time, FAEEs usually predomi-nated, comprising ca. 80– 90% of total FASCAEs, in the fruits of Euonymus speciesstudied earlier [3]. In this work, a high content of FAEEs (ca. 46%) was observed onlyin the immature arils of E. maximowiczianus.

Moreover, the FAEEs of this species were to a great extent similar to FAMEs intheir FA composition (Table 4). Earlier, an analogous similarity between FAMEs and


1) In our previous communication [3], due to a shortage of plant material, these esters wereerroneously presented as FA propyl esters.

Table 2. Relative Retention Times (RRT) on GC of Individual FASCAE from Fruit Arils and Seeds ofCelastraceae Plants

No. FASCAE RRT�pb) [%] No. FASCAE RRT�p [%]

1 Me 14 : 0 0.782�0.18 17 Me 18 : 1 (n�7) 1.287�0.092 i-Pr 14 : 0 0.819�0.05 18 Et 18 : 0 1.300�0.053 Et 14 : 0b) 0.831�0.05 19 i-Pr 18 : 1 (n�9) 1.316�0.164 Me 16 : 0 1.000�0.00 20 Et 18 : 1 (n�9) 1.331�0.175 Me 16 : 1 (n�9) 1.028�0.05 21 Me 18 : 2 (n�6) 1.342�0.116 Me 16 : 1 (n�7) 1.038�0.02 22 i-Pr 18 : 2 (n�6) 1.378�0.097 Me 16 : 1 (n�5) 1.046�0.04 23 Et 18 : 2 (n�6) 1.394�0.188 i-Pr 16 : 0 1.049�0.02 24 Me 18 : 3 (n�3) 1.422�0.079 Et 16 : 0 1.052�0.04 25 i-Pr 18 : 3 (n�3) 1.457�0.07

10 Et 16 : 1 (n�9) 1.065�0.05 26 Et 18 : 3 (n�3) 1.479�0.0411 Et 16 : 1 (n�7) 1.079�0.06 27 Me 20 : 0 1.508�0.0612 Et 16 : 1 (n�5) 1.090�0.11 28 Me 20 : 1 (n�9) 1.550�0.0813 Me 17 : 0 1.120�0.08 29 Bu 18 : 2 (n�6)c) 1.672�0.0814 Me 18 : 0 1.245�0.07 30 Me 22 : 0 1.859�0.0815 Me 18 : 1 (n�9) 1.278�0.07 31 Me 23 : 0 2.099�0.0316 Bu 16 : 0b) 1.281�0.02 32 Me 24 : 0 2.392�0.02

a) FA Designations: 14 : 0, myristic; 16 : 0, palmitic; 16 : 1 (n�9), (Z)-hexadec-7-enoic; 16 : 1 (n�7),palmitoleic; 16 : 1 (n�5), palmitovaccenic; 17 : 0, margaric; 18 : 0, stearic; 18 : 1 (n�9), oleic; 18 : 1(n�7), (Z)-vaccenic; 18 : 2 (n�6), linoleic; 18 : 3 (n�3), a-linolenic; 20 : 0, arachidic; 20 : 1 (n�9),gondoic; 22 : 0, behenic; 23 : 0, tricosanoic; 24 : 0, lignoceric. b) p [%], Relative standard deviation.c) Calculated from [3].

FAEEs was established in the arils of E. maximowiczianus (mature), E. sacrosanctus,and E. alatus�E. sacrosanctus fruits (see Table 3 in [3]). So, it can be suggested that, inthe arils from these species, FAMEs and FAEEs were formed at the expense of a sameFA pool characteristic for a given plant species. However, in other species and sections,no such similarity between FAMEs and FAEEs in their FA composition was observed.Thus, no particular section of the Euonymus genus could be characterized by thesimilarity of such indices as UI (unsaturation index) values, qualitative and quantitativecomposition of various FASCAE fractions, their FA composition, etc.

An unusual FASCAE composition was observed also in C. rugosus, where theyconsisted exclusively of FAMEs, and in E. alatus (see below).

The FAIPEs occurred in the arils of eight species and usually ranked below FAMEsin their concentration in total FASCAEs (Table 3). There was only one exception: inthe total FASCAEs of E. alatus, the FAIPEs comprised 86.4% and consisted almostexclusively of isopropyl myristate (Table 4). A FAIPE mixture of about the samecomposition was present also in the seeds of this species (Tables 3 and 5), where thecorresponding value was as high as 94.6%. As shown in Table 4, in all species except E.maximowiczianus, the aril FAIPEs contained only myristic acid, which, in its turn, wasrarely, if ever, found in other FASCAE fractions. Finally, seed FAIPEs from E.phellomanus and E. alatus (Table 5) were comprised of only isopropyl myristate(Fig. 2).

Earlier [3], only traces of myristate were found (see above), while FAIPEs wererepresented either by 100% isopropyl linoleate (in arils), or by its equimolar mixturewith isopropyl oleate (in seeds). FABEs were also characterized by a very limitedassortment of FAs. In E. sacrosanctus seeds, they contained only linoleic acid, while inE. europaeus arils they were represented by a linoleate/palmitate 7 :3 mixture.


Table 3. Composition of FASCAE Fractions [wt-%] in Fruit Arils and Seeds of Various CelastraceaeSpecies

Plant species Arils Seeds

FAME FAEE FAIPE FAME FAEE FAIPE

Euonymus sectionE. bungeanus 73.5 2.6 23.9 – – –E. europaeusa) 25.6 48.7 11.3 28.5 63.5 7.9E. hamiltonianus 71.3 – 28.7 – – –E. phellomanus 92.2 7.8 – 74.6 10.9 14.5E. sieboldianus 83.7 – 17.3 – – –Melanocarya sectionE. alatus 8.5 5.2 86.4 4.2 1.2 94.6E. sacrosanctusb) 6.8 93.2 – – 41.4 51.8Kalonymus sectionE. latifolius 69.6 6.1 24.3 – – –E. macropterus 66.7 – 33.3 – – –E. maximowiczianus 42.1 45.9 12.0 – – –Axillares sectionCelastrus rugosus 100.0 – – – – –

a) Data from [3]. b) Data from [3]; FASCAEs from seeds contained also FABEs (6.7%).

Special features of FA composition of FAIPEs and FABEs found in this worksuggest that these esters significantly differ from FAMEs and FAEEs in the mechanismof their biosynthesis. In fact, FAMEs and FAEEs were characterized by a muchbroader FA assortment, while their strict selectivity with respect to a certain lower-alcohol or FA molecular species was apparent in only two cases, viz. in C. rugosus (seeabove) and in E. bungeanus (Table 4).

As a whole, as before (see Tables 3 and 4 in [3]), linoleic, palmitic, and oleic acidspredominated in the FA composition of FAMEs and FAEEs from both parts of thefruit. At the same time, a-linolenic acid was present in noticeable concentrations onlyin the arils of Kalonymus section species, while previously its very considerableamounts, as methyl and ethyl linolenate, were found in E. sacrosanctus and E. alatus�E. sacrosanctus (Melanocarya section), and in E. europaeus (Euonymus section).Linoleate levels and, consequently, UI values of these FAMEs and FAEEs were alsomuch lower than those found before.

In addition, the FASCAEs presented in Tables 4 and 5 differed from those studiedearlier (see Tables 3 and 4 in [3]) in the presence of myristic (see above), margaric, andvery-long-chain n-saturated FAs (m¼20 and 22– 24), as well as in an enhancedconcentration of palmitic, oleic, and stearic acids and in a lower amount ofpalmitoleate.


Fig. 2. Mass spectrum of isopropyl myristate from E. phellomanus seeds


Tabl

e4.

FAC

ompo

sitio

nof

FAM

E,F

AE

E,a

ndFA

IPE

Fra

ctio

ns,a

ndT

otal

FASC

AE

sfr

omF

ruit

Ari

lsof

Var

ious

Cel

astr

acea

eSp

ecie

s

FASC

AE

Fra

ctio

nsFa

tty

acid

s(%

ofto

talF

As)

UI

14:0

16:0

16:1

(n�

7)16

:1(n�

5)17

:018

:018

:1(n�

9)18

:1(n�

7)18

:2(n�

6)18

:3(n�

3)20

:022

:024

:0

E.b

unge

anus

FAM

E–

35.7

––

–6.

042

.8–

15.5

––

––

0.73

8FA

EE

––

––

––

–10

0.0

––

––

2.00

0FA

IPE

100.

0–

––

––

––

––

––

–0

Tota

lFA

SCA

Es

23.9

26.2

––

–4.

431

.5–

14.0

––

––

0.59

5

E.h

amilt

onia

nus

FAM

E–

51.7

––

–5.

229

.1–

11.1

–1.

61.

3–

0.51

3FA

IPE

100.

0–

––

––

––

––

––

–0

Tota

lFA

SCA

Es

28.7

36.8

––

–3.

720

.8–

7.9

–1.

20.

9–

0.36

6

E.p

hello

man

usFA

ME

3.5

32.6

3.3

–1.

06.

328

.92.

816

.62.

11.

01.

40.

50.

745

FAE

E–

40.0

––

––

31.9

–28

.1–

––

–0.

881

Tota

lFA

SCA

Es

3.3

33.5

2.1

–0.

95.

929

.52.

617

.61.

90.

91.

30.

50.

751

E.s

iebo

ldia

nus

FAM

E–

28.1

––

5.4

2.2

31.3

1.6

24.6

6.7

––

–1.

022

FAIP

E10

0.0

––

––

––

––

––

––

0To

tal

FASC

AE

s17

.323

.1–

–4.

51.

826

.21.

320

.25.

6–

––

0.86

0

E.a

latu

sa )FA

ME

–23

.8–

––

8.3

33.1

–18

.0–

––

11.4

0.69

1FA

EE

–25

.1–

––

––

–27

.247

.8–

––

1.97

8FA

IPE

99.0

1.0

––

––

––

––

––

–0

Tota

lFA

SCA

Es

85.5

4.2

––

–0.

72.

8–

2.9

2.5

––

1.0

0.16

1

E.l

atif

oliu

sb)

FAM

E–

22.4

––

–2.

716

.91.

126

.327

.01.

9–

0.9

1.51

6FA

EE

––

––

––

35.8

–35

.129

.1–

––

0.93

3FA

IPE

100.

0–

––

––

––

––

––

–0

Tota

lFA

SCA

Es

24.4

15.6

––

–1.

921

.80.

818

.318

.81.

3–

0.6

1.05

6


Tab

le4

(con

t.)

FASC

AE

Fra

ctio

nsFa

tty

acid

s(%

ofto

talF

As)

UI

14:0

16:0

16:1

(n�

7)16

:1(n�

5)17

:018

:018

:1(n�

9)18

:1(n�

7)18

:2(n�

6)18

:3(n�

3)20

:022

:024

:0

E.m

acro

pter

usFA

ME

–38

.9–

––

7.1

31.8

–11

.510

.7–

––

0.86

9FA

IPE

100.

0–

––

––

––

––

––

–0

Tota

lFA

SCA

Es

33.3

26.0

––

–4.

721

.2–

7.7

7.1

––

–0.

579

E.m

axim

owic

zian

usc )

FAM

E–

8.1

1.9

2.6

–0.

920

.45.

552

.25.

9–

––

1.43

0FA

EE

–3.

42.

52.

1–

0.7

24.5

–61

.25.

5–

––

1.55

0FA

IPE

23.0

––

––

–18

.0–

52.0

7.0

––

–1.

681

Tota

lFA

SCA

Es

2.8

5.0

2.0

2.0

–0.

722

.02.

356

.35.

9–

––

1.59

6

Cel

astr

usru

gosu

sFA

ME

–9.

1–

––

3.5

35.3

0.8

50.2

–0.

20.

40.

11.

373

a )FA

ME

san

dto

talF

ASC

AE

sco

ntai

ned

also

tric

osan

oic

acid

(5.4

and

0.5%

,res

p.).

b)

FAM

Es

and

tota

lFA

SCA

Es

cont

aine

dal

sotr

icos

anoi

cac

id(0

.8an

d0.

5%,r

esp.

).c )

FAM

Es

and

tota

lFA

SCA

Es

cont

aine

dal

so16

:1(n�

9)an

d20

:1(n�

9)ac

ids

(0.7

and

0.3,

and

1.8

and

0.7%

,res

p.),

and

FAE

Es

cont

aine

dal

so16

:1(n�

9)ac

id(0

.1%

).

Present results, as well as previous ones [3], indicate that, in Celastraceae plants, theFASCAEs are formed mostly in the arils (see above), which, moreover, greatly exceedthe seeds in a much broader assortment of FAs in the FASCAEs indicating that the arilswere much more active in the biosynthesis of FASCAEs than the seeds.

The molecular mechanism of this biosynthesis in higher plants is still unclear, andfurther investigations are necessary. However, some conjectures on this problem can beattained from our data on the FA composition of FASCAEs in the fruits ofCelastraceae plants. So, based on the evidence presented in Tables 3 – 5, it can besuggested, first, that, in the arils from some species, FAMEs and FAEEs could beformed at the expense of a same FA pool and, second, that FAIPE and FABEs couldsignificantly differ from FAMEs and FAEEs in the mechanism of their biosynthesis.

For further evidence (see also [3]), we have compared the FASCAEs from arils andseeds (Tables 4 and 5) with the triacylglycerols (TAGs) of the same origin (Table 6) asregards their FA compositions; the TAGs considerably exceeded FASCAEs in theirconcentration in fruit lipids. It can be seen that, in both fruit parts, linoleic, palmitic, andoleic acids predominated both in the TAGs and in the FAMEs and FAEEs (see above),and E. europaeus lipids were always characterized by a highest UI. At the same time,we did not find any significant indices of correlation between total FASCAEs andTAGs within a given plant species with respect to their FA composition. As an example(Table 6), we refer to the aril TAGs of E. sieboldianus, which were characterized by anincreased level of palmitoleic acid, and of E. bungeanus, which included 6.7% of 16 :1(n�8) FA and 2.3% of 18 :1 (n�8) FA; in fact, none of these unusual FAs werepresent in the total FASCAEs of respective plant species (Table 4). Thus, FASCAEsand TAGs of fruits could scarcely be formed at the expense of a same FA pool.Moreover, the same conclusion must be reacted as regards separate FASCAE fractionspresent in the same fruit part of a given plant species, because these fractions almostwithout exception, considerably differed from one another with respect to their FAcomposition.

Occurrence of FASCAEs in other Plants and Fungi. While investigating the lipidcomposition of plant and fungi, other groups have discovered FASCAEs in the neutrallipids of corn pollen [8], walnut fruit oil [9], dry needles of fir and spruce [10], callus


Table 5. Fatty-Acid Composition of FASCAE Fractions from Seeds of Various Euonymus Species

Euonymus FASCAE Fatty acids (% of total FA) UIspecies fractions 14 : 0 16 : 0 18 : 0 18 : 1(n�9) 18 : 2 (n�6) 18 : 3 (n�3)

E. phellomanus FAME – 14.1 5.5 36.9 43.5 – 1.239FAEE – 15.4 – 84.6 – – 0.846FAIPE 100.0 – – – – – 0Total FASCAEs 14.5 12.2 4.1 36.7 32.5 – 1.017

E. alatusa) FAME – 45.8 4.1 23.5 19.2 2.3 0.687FAEE – 41.5 – 20.5 38.0 – 0.966FAIPE 100.0 – – – – – 0Total FASCAEs 94.6 2.4 0.2 1.2 1.3 0.1 0.041

a) FAMEs and total FASCAEs contained also margaric acid (5.1 and 0.2%, resp.).


Tabl

e6.

Fatty

Aci

dC

ompo

sitio

nof

Tri

acyl

glyc

erol

s(T

AG

s)in

Fru

itA

rils

and

Seed

sof

Var

ious

Cel

astr

acea

eSp

ecie

sa )

Pla

ntsp

ecie

sFa

tty

acid

([%

]of

tota

lFA

)U

I

16:0

16:1

(n�

7)16

:1(n�

5)18

:018

:1(n�

9)18

:1(n�

7)18

:2(n�

6)18

:3(n�

3)

Ari

lsa )

E.b

unge

anus

b)

29.3

3.9

–1.

411

.23.

039

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culture from mint leaves [11], dry rhizomes of ginseng [12], dry matter of liverwort[13], and mycelium of a filamentous fungus Rhizopus arrhizus [14] . The FASCAEswere represented mostly by FAMEs and, only in two cases [13] [14], by FAEEs. Theconcentrations of FAMEs and FAEEs were low in almost all instances (<1%). As inour work, the FAMEs found in plants did not represent artifacts of the experiment (seeabove). As in the FASCAEs from Celastraceae (Tables 4 and 5), saturated andunsaturated FAs with m�18 predominated among the FAs, and FAs with m>18,typical for wax esters, were found only in the FAMEs from fir and spruce needles [10].In the rhizomes of ginseng, the FASCAEs were represented only by methyl palmitate[12], and the FAMEs from mint callus culture were similar to its TAGs in their FAcomposition [11].

Small amounts of FASCAEs were also found in the volatile compounds of suchplant sources as the leaves of some Rutaceae species (Ruta graveolens, Haplophyllumsuaveolens, Zanthoxylum limoncello, Z. panamense and Z. setulosum) [15], fruits ofmango (Mangifera indica L.) [16] and Mandragora autumnalis L. [17], leaves and fruitsof Ficus carica L. [18], �maari�, a fermented product from baobab (Adansonia digitataL.) seeds [19], as well as plum (Prunus domestica L.) fruits [20].

In these experiments, as distinct from our work (see Exper. Part), there were nostorage of plant materials at �188 and their subsequent fixation with boiling water;instead, the materials were either directly homogenized in water [16] [17] [20], or air-dried [17]. In one case [16], to isolate volatile compounds from fruits, a prolongedEtOH extraction was used; as a result, only the FAEEs were found as FASCAEs thusobtained. In other instances, steam-distillation�extraction in vacuo was used to attainthis end. FAMEs and FAEEs predominated in the FASCAEs of the investigated plants.They included myristic, palmitic, oleic, linoleic, and a-linolenic acids. Also present weremethyl and ethyl palmitoleate [15], methyl and ethyl stearate [15] [16] [20], FAIPEs[16] [17] [20], and FABEs [16] [19] [20]. FA Esters of other short-chain alcohols werefound in mango fruits [16]. Taking into account the techniques employed by theseworkers for isolating volatile-compound mixtures, one can suggest that manyFASCAEs found in these mixtures could represent artifacts of the experiment.

Meanwhile, noticeable amounts of FASCAEs were recently found in the greenmicroalga Chlamydomonas reinhardtii. These esters included five FAMEs and threeFAEEs, which contained primarily palmitic, oleic, and linoleic acid residues. TheFASCAEs were natural products, because they were extracted from a freeze-driedmaterial with a MeOH-free hexane, and they are of interest due to their potential use asbiofuels [21]. Brief mention should also be made of the recent review by Zhu et al. [22],in which more than 200 chemical constituents from ca. 61 Euonymus species werereported, but no FASCAEs except ricinoleic acid methyl ester were mentioned.

Conclusions. – The FASCAEs are present in most plants studied in this work andrepresent a wide variety of species, which include the residues of several individual FAsand several short-chain alcohols. The composition of various FASCAE fractions variesconsiderably depending on the taxonomic position of a plant species, fruit part, theextent of fruit maturity, etc. Their formation in the fruit arils proceeds by far moreefficiently than in the seeds. All these esters are natural products of cell biosynthesisrather than artifacts of the experiment. The FASCAEs and TAGs of fruits could


scarcely be formed at the expense of a same FA pool, and the same conclusion must bedrawn regarding separate FASCAE fractions present in the same fruit part of a givenplant species. Certain preliminary conjectures on the molecular mechanism ofFASCAE biosynthesis in higher plants can be made on the basis of our data on theFA composition of FASCAEs in Celastraceae. However, as a whole, this mechanism isstill unclear, and further investigations are necessary for its unveiling, especiallyconsidering a potential role of FASCAEs as biofuels.

This work was partly supported by the Russian Foundation for Basic Research (grant No. 12-04-31850). We are grateful to Dr. N. A. Trusov, Main Botanical Garden of Russian Academy of Sciences, forhis help in obtaining plant material and the picture of an E. maximowiczianus fruit.

Experimental Part

Abbreviations: FA, fatty acid, FAEE, FA ethyl ester; FAIPE, FA isopropyl ester; FAME, FA methylester; FASCAE, FA short-chain-alkyl ester. FA designations: 14 : 0, myristic; 16 : 0, palmitic; 16 : 1 (n�9), (Z)-hexadec-7-enoic; 16 : 1 (n�7), palmitoleic; 16 : 1 (n�5), palmitovaccenic; 17 : 0, margaric; 18 :0,stearic; 18 : 1 (n�9), oleic; 18 : 1 (n�7), (Z)-vaccenic; 18 : 2 (n�6), linoleic; 18 : 3 (n�3), a-linolenic;20 : 0, arachidic; 20 : 1 (n�9), gondoic; 22 : 0, behenic; 23 : 0, tricosanoic; 24 : 0, lignoceric; m, number ofC-atoms in an aliphatic chain; Rf, retention factor; RRT, relative retention time; TAG, triacylglycerol;UI, unsaturation index.

Plant Material and Isolation of FASCAEs. Mature fruits of 16 Celastraceae species (Table 1) werecollected during 2009–2011 in the arboretum of the Main Botanical Garden of the Russian Academy ofSciences and stored at �188. Herbarium voucher specimens were deposited with the Herbarium of thisGarden (Moscow, Russia). A sample of fruits (ca. 10 g) was fixed for 1 min with boiling water toinactivate enzymes and to remove a possible admixture of H2O-soluble alcohols. The fruits wereseparated into seeds and H2O-saturated arils. Both parts of the fruit were separately mixed with an equalvolume of H2O and homogenized. Neutral lipids were extracted from the homogenate using freshlypurified CHCl3 devoid of alcohols [7] and stored as a benzene soln. at �188. After removing benzene invacuo, the lipids were dissolved in hexane and fractionated by prep. TLC [5] using a 20�20-cm Kieselgel60F (Merck) plate preimpregnated with a 0.001% 2’,7’-dichlorofluorescein soln., the hexane/Et2O system(95 : 5) serving as a mobile phase. The FASCAE zone (Rf 0.85) was visualized under UV light (l 254 nm)and eluted with hexane.

Analyses of FASCAE Composition. FASCAE Composition was determined by GC/MS using theAgilent 7890A GC (Agilent Technologies, Inc., USA) device [3].

A portion of the initial FASCAE mixture was converted into FAMEs [6], and their FA compositionwas determined. A remainder of the mixture was separated into individual FASCAE species, which werethen quantified and identified. The NIST and Wiley MS search libraries were used for identification, andonly the compounds with a high quality index (degree of agreement between MS of sample and MS in thedatabase >90) were retained. In addition, the MS of all FASCAEs were shown to be identical to thosepresented in the Lipid Library Archive [6].

The data thus obtained were used for calculating the content and FA composition of FAME, FAEE,and FAIPE fractions (wt%).

Triacylglycerols (TAGs) were obtained from the aril and seed CHCl3 extracts by prep. TLC [3] andconverted into FAMEs [5]. The compositions of FAMEs thus obtained and their unsaturation indices(UIs), as well as the UI of total FASCAEs and their fractions were determined as described in [3].

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Debrah, M. Jakobsen, Afr. J. Biotechnol. 2011, 10, 4197.[20] J. A. Pino, C. E. Quijano, Cienc. Technol. Aliment. Campinas 2012, 32, 76.[21] V. A. Herrera-Valencia, R. A. Us-Vazquez, F. A. Larque-Saavedra, L. F. Barahona-Perez, Ann.

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Biodiversity 2012, 9, 1055.

Received September 19, 2012


occurrence of fatty acid short-chain-alkyl esters in fruits of celastraceae plants

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