Novel galactolipids from the leaves of Ipomoea batatas L.: Characterization by liquid chromatography coupled with electrospray ionization-quadrupole time-of-flight tandem mass spectrometry

Article abstract of DOI:10.1021/jf071331z

Sixteen novel and ten known galactolipids have been isolated and characterized from the leaves of Ipomoea batatas L. (sweet potato) using an analytical method based on high-performance liquid chromatography coupled with electrospray ionization-quadrupole

Full text of DOI:10.1021/jf071331z

J. Agric. Food Chem. 2007, 55, 10289–10297 10289
Novel Galactolipids from the Leaves of Ipomoea
batatas L.: Characterization by Liquid
Chromatography Coupled with Electrospray
Ionization–Quadrupole Time-of-Flight Tandem Mass
Spectrometry
ASSUNTA NAPOLITANO,† VIRGINIA CARBONE,‡ PAOLA SAGGESE,‡
KINYA TAKAGAKI,§ AND COSIMO PIZZA*,†
Dipartimento di Scienze Farmaceutiche, Università degli Studi di Salerno, Via Ponte Don Melillo,
84084 Fisciano, Salerno, Italy, Centro di Spettrometria di Massa Proteomica e Biomolecolare, Istituto
di Scienze dell’Alimentazione del Consiglio Nazionale delle Ricerche, Via Roma 52 A-C,
83100 Avellino, Italy, and Toyo Shinyaku, 7-28 Yayoigaoka, Tosu-City, Saga, Japan
Sixteen novel and ten known galactolipids have been isolated and characterized from the leaves of
Ipomoea batatas L. (sweet potato) using an analytical method based on high-performance liquid
chromatography coupled with electrospray ionization–quadrupole time-of-flight tandem mass spec-
trometry. Using this technique, the structures and regiochemistries of the fatty acyl groups and the
positions of the double bonds on the acyl chains were determined. Sugar moieties were identified by
analysis of one- and two-dimensional nuclear magnetic resonance spectra. The positions of the double
bonds of polyunsaturated fatty acids were confirmed, and in some cases their geometries determined,
by gas chromatography–mass spectrometry. This is the first report of galactolipids in the leaves of
sweet potato.
KEYWORDS: Monogalactosyldiacylglycerol (MGDG); digalactosyldiacylglycerol (DGDG); tandem mass
spectrometry; nuclear magnetic resonance spectroscopy; sweet potato; Ipomoea batatas L.
INTRODUCTION
presenting carcinostatic activities. The glycolipid fraction of the
plant material was shown to suppress proliferation and to
promote differentiation of cancer cells and was hence claimed
to be responsible for the carcinostatic properties described (5).
Ipomoea batatas (L.) Lam. (Convolvulaceae), commonly
known as sweet potato, is a tuberous-rooted herbaceous species
cultivated throughout the world for its edible tubers that are
either consumed raw or after cooking (1). Additionally, the
leaves of I. batatas are an excellent source of bioactive
anthocyanin and polyphenolic constituents (2) and may be used
as a fresh vegetable or in the form of tea, noodles, bread, or
confectionery. A recent study concerning the nutritional com-
position and physiological functions of the leaves suggested that
the sweet potato could represent a beneficial food source by
virtue of the presence of polyphenolic compounds (2). In this
context, 15 anthocyanins and 6 polyphenolics have been
identified and quantified in I. batatas (2), and the leaves are
reportedlyrichinvitaminB,iron,calcium,zinc,andproteins(3,4).
According to a patent filed by Osamu et al. (5), some parts
of sweet potato, especially the leaves, runners, stalks, and
rhizomes, can be employed as functional foods or drinks
Glycolipids are the major constituents of plastid membranes
and constitute up to 80% of thylakoid membrane glycerolipids,
approximately 50% of which are comprised of monogalacto-
syldiacylglycerols (MGDG) (6). Various studies have shown
that glycoglycerolipids exhibit specific biological properties
including antiviral (7), antitumor (8–13), and antiinflammatory
(14) activities. Additionally, they appear to play a role in the
inhibition (15) and promotion (16, 17) of cell growth and in
protection against cell death (8–13, 18).
The objective of the present study was, therefore, the struc-
tural characterization of the galactolipids comprising the nonpolar
fraction derived from leaves of I. batatas. In the structures of
the digalactosyldiacylglycerol (DGDG) and MGDG species
present in this fraction, two fatty acyl groups are attached at
the sn-1 and sn-2 positions of the glycerol backbone, and one
or two sugar moieties are linked to the sn-3 position of the glyc-
erol via acetal linkage. In order to achieve a complete structural
characterization of these galactolipids, including the regiochem-
istries of the fatty acyl groups and the positions of the double
bonds on the acyl chains, an analytical method based on high-
* Corresponding author. Tel: +39-089969765. Fax: +39-089969602.
E-mail: pizza@unisa.it.
† Università degli Studi di Salerno.
‡ Istituto di Scienze dell’Alimentazione del Consiglio Nazionale delle
Ricerche.
§ Toyo Shinyaku.
10.1021/jf071331z CCC: $37.00
2007 American Chemical Society
Published on Web 11/08/2007

10290 J. Agric. Food Chem., Vol. 55, No. 25, 2007
Napolitano et al.
MHz spectrometer at 300 K with samples dissolved in CD3OD (99.96%
D; Sigma Aldrich, Milan, Italy) and calibrated using the solvent signal
as internal standard (1H, δ ) 3.34 ppm; 13C, δ ) 49.0 ppm).
Preparation of Fatty Acid Methyl Esters. Galactolipids, previously
purified by HPLC, were subjected to direct transesterification by heating
at 80 °C for 4 h in glass tubes together with 2.5 mL of 2% sulfuric
acid in methanol. After reaction, the tubes were allowed to cool, 2.0
mL of water and 1.0 mL of n-hexane were added, and the tubes were
sealed and vortex-mixed for 10 min. The tubes were centrifuged for 1
min at 4000 rpm in order to accelerate phase separation, and the
n-hexane phase was transferred to GC vials. A further volume (1.0
mL) of n-hexane was added to the aqueous phase, and a further
extraction was carried out as described above: this step was repeated
twice.
GC-MS Analyses. Fatty acid methyl esters were analyzed using a
Waters GCT orthogonal acceleration time-of-flight mass spectrometer
coupled with an Agilent Technologies (Palo Alto, CA) GC6890 series
GC system fitted with a DB-1 capillary column (30 m × 0.25 mm i.d;
0.25 µm film thickness; J&W Scientific, Folsom, CA). The carrier gas
was helium at a flow rate of 1 mL/min, sample injection was performed
in the splitless mode with an injector temperature of 280 °C, the ion
source temperature was 200 °C, and the GC-MS interface temperature
was 300 °C. The oven temperature was initially held at 40 °C for 5
min, then increased to 60 °C at 4 °C/min, to 150 °C at 15 °C/min, to
280 °C at 3 °C/min, and finally to 300 °C at 20 °C/min; the total run
time was 60 min. Mass spectra were recorded at 70 eV ionization energy
in the 50–500 mass range with a scan time of 0.5 s.
performance liquid chromatography coupled with electrospray
ionization–quadrupole time-of-flight tandem mass spectrometry
(HPLC-ESI/Q-TOF/MS/MS) was developed. The sugar units
and their configurations were assigned using spectroscopic
methods including the concerted application of one- and two-
1
dimensional NMR techniques (13C NMR, H NMR, selective
1D-TOCSY, HSQC, and HMBC). Additionally, the positions
of the double bonds of the polyunsaturated fatty acids attached
to the two carbons of the glycerol galactolipid backbones were
confirmed, and in some cases their geometries determined, by
gas chromatography–mass spectrometric (GC-MS) analysis of
the derivatized fatty acids. Using the described strategy, 16 new
(1, 6–14, 17, 22–26) and 10 known galactolipids were isolated
and characterized. To the best of our knowledge, this is the
first report of galactolipids in the leaves of sweet potato.
MATERIALS AND METHODS
Chemicals. HPLC grade methanol was purchased from J. T. Baker
(Baker Mallinckrodt, Phillipsburg, NJ), and acetonitrile and formic acid
were obtained from Carlo Erba (Milan, Italy). HPLC grade water (18
mΩ) was prepared using a Millipore (Bedford, MA) Milli-Q purification
system.
Preparation of Samples. I. batatas (L.) Lam. (cultivar Suioh) was
cultivated at the National Agricultural Research Center for the Kyusyu
Okinawa Region, Japan, and a voucher specimen deposited in the
herbarium at the Research Center. Fresh leaves were harvested in July
2004 and were washed, air-dried, powdered, and stored at room
temperature until required for analysis.
RESULTS
HPLC-UV Analyses. In order to optimize chromatographic
conditions, and to isolate pure compounds for NMR and GC-
MS analyses, a preliminary HPLC-UV separation was carried
out on the nonpolar fraction of the leaf extract of I. batatas
eluted from the C18 Sep-Pak cartridge. While HPLC with
elution method A (as described in Materials and Methods) gave
a good separation of the more nonpolar compounds, it was not
appropriate for separating the less nonpolar components. For
this reason, elution method B, employing a different ratio of
organic solvent to water in the mobile phase, was developed.
Employing these elution methods, a total of 26 compounds were
separated and collected, 16 being obtained with elution method
A and 10 with elution method B (see Tables 1 and 2).
NMR Analyses. The occurrence of glycerolipid structures
was established from the 1H NMR spectra of the purified
compounds in which the proton signals associated with H-2 of
the D-glycerol units exhibited a characteristic resonance at δ
5.29 indicating a C-2 linkage to an O-acyl group (19). Further
analyses of the sugar regions of the spectra indicated that the
compounds were galactolipids, and two groups of molecules
(i.e., MGDG and DGDG) could be identified differing only in
the number of galactopyranose units O-linked to the di-O-
acylglycerol backbone.
A sample (3 g) of the powdered leaf material was extracted with 60
mL of methanol for 8 days at room temperature. The extract was filtered
and the solvent removed to yield 320 mg of crude extract. In order to
separate the nonpolar components from the polyphenols and antho-
cyanins, a methanolic solution of the extract was passed through a C18
Sep-Pak cartridge (Waters Corp., Milford, MA) that had been precon-
ditioned with methanol, water, and 0.2% formic acid. After being
washed with 0.2% formic acid and 0.2% formic acid–methanol (50:50
v/v), the nonpolar components were eluted with methanol, and the
methanolic fraction (240 mg) was retained for chromatographic
analyses.
HPLC-UV Analyses. The methanolic fraction from the C18 Sep-
Pak separation was analyzed by HPLC using a Waters Alliance 2695
liquid chromatograph equipped with a Dual Lambda absorbance
detector. Individual galactolipids were eluted isocratically from a
Thermo (Bellefonte, PA) Hypersil BDS C18 column (250 × 4.6 mm
i.d.; 5 µm) with a mobile phase of methanol–water–acetonitrile (90.5:
7:2.5) (elution method A) at a flow rate of 1 mL/min and with detection
at 205 nm. In order to improve the separation of the more polar
galactolipids, a mobile phase of methanol–water–acetonitrile (82.5:15:2.5)
(elution method B) was also employed.
HPLC-ESI/MS and HPLC-ESI/MS/MS Analyses. The methanolic
fraction from the C18 Sep-Pak separation was analyzed by HPLC-
ESI/MS “on-line” using a Waters Micro-Hybrid quadrupole orthogonal
acceleration time-of-flight mass spectrometer coupled with a Waters
Alliance 2695 HPLC through a Waters orthogonal Z-spray source
interface. Individual galactolipids were separated using the chromato-
graphic conditions described above. The eluate was directly injected
into the electrospray ion source, and the MS1 and MS/MS spectra were
acquired in the positive ion mode and interpreted using the Mass Lynx
4.0 software provided by the manufacturer. The desolvation temperature
was 180 °C, the source temperature was 100 °C, the capillary voltage was
3400 V, the sample cone voltage was 40 V, the extraction voltage was
1.5 V, and the collision cell voltage was 7 V. MS/MS spectra were
obtained in the survey scan mode, using a range of collision energies
between 30 and 45 V, and recorded in the m/z 50–1500 region.
Galactolipids were quantified by HPLC-ESI/MS using an external
standard calibration method. Samples were analyzed in triplicate and
the mean and standard deviation values reported.
With respect to the first group of molecules, analysis of the
1H NMR and selective 1D-TOCSY spectra revealed the presence
of one ꢀ-D-galactopyranosyl unit. ꢀ-Glycosylation of the
glycerol moiety was confirmed by the low- and high-field
chemical shifts exhibited by the anomeric carbon C1′ (δ 105.0)
and proton H1′ (δ 4.26), respectively, as well as by the large
3JH1′–H2′ coupling constant (J ) 7 Hz). Moreover, the small
coupling constant of the proton H4′ (δ 3.86, J ) 2.5 and 4.0
Hz) in the sugar residue, together with the large value of the
3JH2′–H3′ coupling constant (J ) 9 Hz), verified that the sugar
unit was galactose.
In the case of the second group of molecules, examination
of the anomeric region of the HSQC spectrum showed that the
saccharide portion consisted of two sugars. The selective 1D-
NMR Analyses. The 1H and 13C NMR spectra were recorded on a
Bruker (Bruker BioSpin AG, Fällanden, Switzerland) model DRX-600

Novel Galactolipids from the Leaves of I. batatas L.
J. Agric. Food Chem., Vol. 55, No. 25, 2007 10291
Table 1. Fatty Acid Composition and Quantitative Amounts of MGDG Compounds Isolated from I. batatas Leaves
a According to elution method A. b Novel galactolipid. c According to elution method B. * The results are reported as mean ( standard deviation.
Table 2. Fatty Acid Composition and Quantitative Amounts of DGDG Compounds Isolated from I. batatas Leaves
a According to elution method A. b Novel galactolipid. c According to elution method B. * The results are reported as mean ( standard deviation.
TOCSY spectra indicated the presence of one R-D-galactopy-
ranosyl unit and one ꢀ-D-galactopyranosyl unit. The anomeric
signal at δ 4.28 (J ) 7.3 Hz), together with the proton signals
of H4′ at δ 3.91 (J ) 2.5 and 4.0 Hz) and of H3′ at δ 3.53 (J
) 9 Hz), established that the ꢀ-galactopyranose sugar was linked
to the D-glycerol moiety. The anomeric signal at δ 4.90 (J )
3.6 Hz), together with the proton signals of H2′′ at δ 3.82
(C18:2 n-6, 9 cis), R-linolenic (C18:3 n-3, 6, 9 cis), nonadeca-
dienoic (C19:2), arachidic (C20:0), 11-eicosenoic (C20:1 n-9
trans), and heneicosanoic (C21:0) acids, and the distribution
of these acids in each HPLC fraction is reported in Tables 1
and 2. The identities of all fatty acids (except for C19:2) were
confirmed by comparison of their retention times with those of
corresponding standards, and in some cases it was possible to
define the geometries of the double bonds in the polyunsaturated
fatty acids. The presence of nonadecadienoic acid (C19:2) was
confirmed on the basis of molecular weight and the MS
fragmentation pattern (Figure 1), which indicated the presence
of double bonds at C-10 and C-13 of the acyl chain.
3
(3JH1′′–H2′′ ) 3.6 Hz, JH2′′–H3′′ ) 9.5 Hz) and of H4′′ at δ 3.94
(J ) 2.5 and 4.0 Hz), implied the presence of an R-galactopy-
ranose sugar. Finally, the HMBC spectrum revealed the crucial
correlation between C1′′ of the external R-galactose and the
geminal H-6′ protons of ꢀ-galactopyranose, thus establishing a
(1′′f6′)-O-glycosidic linkage.
Within each group of galactolipids, the individual components
differed only with respect to the length of the acyl chains of
the fatty acids, their degree of unsaturation, and the positions
of the double bonds. These structural features were not
immediately definable through examination of the NMR data,
and hence more detailed analyses were performed in order
unambiguously to identify each galactolipid and to determine
the regiochemical distribution of the acyl chains.
HPLC-ESI/MS and HPLC-ESI/MS/MS Analyses. The meth-
anolic fraction from the C18 Sep-Pak cartridge was subjected
to HPLC-ESI/MS analysis in order to determine the definitive
structure of each galactolipid. Elution systems with different
percentages of organic phase (methods A and B as described
in Materials and Methods) were used to separate galactolipids
with diverse polarities. Figure 2 presents the HPLC-ESI/MS
profile of the nonpolar fraction from leaves of I. batatas obtained
using elution method A.
GC-MS Analyses of Fatty Acids. With the aim of identifying
the fatty acids linked to the glycerol backbone, the pure
galactolipids previously isolated by HPLC were subjected to
direct transesterification, and the fatty acid methyl esters were
analyzed by GC-MS. The results indicated the presence of
palmitic (C16:0), stearic (C18:0), oleic (C18:1 n-9 cis), linoleic
Analyses were carried out in the presence or absence of 0.2%
formic acid in the mobile phases in order to investigate the effect
of acidic conditions on the separation of the galactolipids and
on their behavior in the electrospray source. The positive ion
HPLC-ESI/MS of the galactolipid-enriched methanolic fraction
showed different MS profiles according to the acidic strength

10292 J. Agric. Food Chem., Vol. 55, No. 25, 2007
Napolitano et al.
Figure 1. Electron impact mass spectrum of methyl nonadecadienoate (m/z 308) derived from compound 22 by transesterification.
Figure 2. HPLC-ESI/MS analysis (using elution method A) of the methanolic extract of leaves of I. batatas. The more polar galactolipids (compounds
6, 7, 11–14, 22, 24–26) were eluted in the first 13 min.
of the eluent employed. Mass spectra acquired following
acidic elution displayed, in addition to [M + Na]+ ions,
fragment ions at [M + H – 162]+ and [M + H – 180]+
associated with the preferred loss of a galactosyl moiety.
Additionally, in-source collision activation of these species
produced further fragmentation arising from the neutral loss
of a fatty acyl group as the free fatty acid from the
diacylglycerol structure, formally represented as [RxCO +
74]+ ions. Under the experimental conditions employed,
simple [RxCO]+ ions were also present in which R1 and R2
indicate the fatty acyl groups linked at the sn-1 and sn-2
positions of the glycerol backbone, respectively. In contrast,
spontaneous in-source fragmentation was not preferred under
nonacidic conditions, and the mass spectra displayed only
[M + Na]+ ions with minor signals originating from [M +
H – 162]+ and [M + H – 180]+ fragments.

Novel Galactolipids from the Leaves of I. batatas L.
J. Agric. Food Chem., Vol. 55, No. 25, 2007 10293
These results confirmed the fatty acid composition of each
galactolipid, as already established by GC-MS analyses, and
indicated variously the presence of C16:0 (palmitic or hexade-
canoic acid), C18:0 (stearic or octadecanoic acid), C18:1
(octadecenoic acid), C18:2 (octadecadienoic acid), C18:3 (oc-
tadecatrienoic acid), C19:2 (nonadecadienoic acid), C20:0
(arachidic or eicosanoic acid), C20:1 (eicosenoic acid), and
C21:0 (heneicosanoic acid). Unfortunately, these analyses did
not allow the regiochemical distribution of each fatty acid to
be established.
from the galactosylglycerol backbone. In addition, fragment ions
at m/z 261.3, 293.2, and 335.4, previously indicated as [R2CO]+,
[R1CO]+, and [R2CO + 74]+ product ions, were also present.
The sodium adduct ions at m/z 301.3 (2D1,2) and 315.3 (3D1,2
)
were formed by the simultaneous neutral losses of one fatty
acid and a part of the other fatty acid, respectively, according
to the fragmentation pathways reported in Figure 3.
Information about the position of the double bonds on the
fatty acyl chains could be obtained from the analysis of the
high-mass region of the MS/MS spectrum. In the typical
fragmentation pathway of a saturated fatty acyl chain, only ions
arising from the neutral losses of CnH2n and CnH2n+2 from the
parent ion can be detected, with the neighboring peaks in the
series being separated by 14 units. However, for unsaturated
acyl chains, neutral losses of CnH2n–2 are also reported, while
the presence of a double bond in the chain reduces the
neighboring peak separation to 12 units (21). In the case of
compound 11, detailed analysis of the high-mass region of the
MS/MS spectrum of the [M + Na]+ ion at m/z 829.9 revealed
a peak at m/z 811.9 associated with the [M + Na – 18]+ ion
and two other peaks at m/z 783.8 and 771.8 resulting from the
above-mentioned losses. The latter two peaks indicated that the
first double bond was located at C-15 of the sn-2 fatty acyl
chain, in agreement with data obtained from the GC-MS
analyses. From a consideration of the separation (i.e., 12 or 14
units) between neighboring peaks in the mass range from m/z
771.8 to m/z 551.5, it was possible to locate the double bonds
on the sn-2 acyl group at C-9 and C-12, confirming the identity
of 9,12,15-octadecatrienoic acid, and the double bond on the
sn-1 acyl chain at C-11, confirming the presence of 11-
eicosenoic acid, both assignments being in agreement with GC-
MS analysis.
The positional distribution of the acyl chains in the galacto-
lipids could be determined from the intensities of the peaks
produced by the loss of carboxylic acids linked to the glycerol
backbone, in agreement with previous literature. Thus, Guella
et al. (20) reported that the loss of the carboxylic acid linked to
the sn-1 glycerol position always produced a more intense peak
than that derived from the loss of the sn-2-linked acyl chain. In
the present case, consideration of the relative intensities of the
[M + Na – R1CO2H]+ and the [M + Na – R2CO2H]+ fragments
derived from the [M + Na]+ precursor ion in the HPLC/ESI/
MS/MS experiments enabled the regiochemical distribution of
the acyl chains to be determined and the galactolipids occurring
in the mixture to be identified (Tables 1 and 2).
The HPLC-ESI/MS/MS data recorded for these compounds
under the analytical conditions described in Materials and
Methods showed a preferred pathway of fragmentation leading
to the formation of [M + Na – 162]+ ions together with product
ions originating from the fragmentation of the galactose moiety.
Moreover, since DGDG compounds contained two galactosyl
units, product ions associated with the loss of the second sugar
from [M + Na – 162]+ ions were also detectable in the MS/
MS spectra. Additionally, spectra obtained under neutral condi-
tions from both groups of galactolipid displayed primarily the
loss of the neutral fatty acid giving rise to [M + Na – R1CO2H]+
and [M + Na – R2CO2H]+ fragments.
For compound 24, which eluted (method B) at a retention
time of 53.08 min, the HPLC-ESI/MS exhibited a peak at m/z
991.9 corresponding to the sodium adduct of a DGDG com-
pound. This component presented the same pattern of fatty acids
as the MGDG species analyzed above, but with two galactosyl
units linked to the sn-3 position of the glycerol backbone.
Moreover, the MS/MS spectrum of 24 (Figure 4) showed a
fragmentation pattern that was very similar to that described
for the associated MGDG. The relative abundance of the two
product ions, [M + Na – R1CO2H]+ and [M + Na – R2CO2H]+,
at m/z 681.7 (G1) and 713.7 (G2), respectively, was informative
about the regiochemical distribution and the composition of the
two fatty acids present in the molecule. It was concluded that
this DGDG presented a 20:1 acyl chain at the C-1 glycerol
position and an 18:3 acyl chain at the C-2 position. A detailed
study of the fragmentation pattern in the high-mass region
verified the presence of one double bond at C-11 of the sn-1
fatty acyl chain, establishing the presence of 11-eicosenoic acid,
and of three double bonds at C-9, C-12, and C-15 of the sn-2
acyl chain, corresponding to 9,12,15-octadecatrienoic acid, both
assignments being in agreement with GC-MS analysis.
Determination of the degree of unsaturation and of the
position of the double bonds in the fatty acids on the galactolipid
acyl chains was achieved by analysis of the MS/MS spectra in
the high-mass regions. In general, for the higher series of MGDG
and DGDG compounds, i.e., those with at least one C20:0, C20:1,
or C21:0 fatty acid linked to carbons of the glycerol backbone,
more complex MS/MS spectra were obtained.
In the case of compound 11, which eluted (method B) at a
retention time of 71.59 min, the HPLC-ESI/MS exhibited a peak
at m/z 829.9 corresponding to the sodium adduct of an MGDG
containing eicosenoic (C20:1) and linolenic (C18:3) acids linked
to the glycerol backbone. In the corresponding MS/MS spectrum
(Figure 3), two abundant product ions were observed at m/z
519.6 and 551.5, referred to as G1 and G2, respectively,
according to the nomenclature used by Kim et al. (21). These
ions resulted from the neutral loss of the fatty acids, C20:1 and
C18:3, respectively (Figure 3), thus allowing the fatty acid
composition to be defined. The regiochemistry of the MGDG
molecule could also be established from the relative intensities
of the [M + Na – RxCO2H]+ ions, on the basis of which the
sn-1 and sn-2 positions were ascribed to the eicosenoic (C20:1)
and linolenic (C18:3) fatty acyl chains, respectively.
In agreement with a previous report (21), fragment ions
originating from the sugar moiety were detected in the low-
mass region. Thus, cleavage of the glycosidic bond generated
the sodium adduct ions at m/z 185.1 (B) and 203.1 (C), in which
the sugar ring lost or retained the hydroxyl group, respectively
(Figure 3). The fragment ion at m/z 243.2 (E) was correlated
with the concomitant cleavage of the two neutral fatty acids
The presence in the MS/MS spectrum of 24 of two fragment
ions at m/z 829.9 and 667.7, originating from two sequential
neutral losses of 162 units associated with each galactose moiety,
verified that the structure of DGDG was related to that of
MGDG 11. Analysis of the low-mass region of the MS/MS
spectrum of 24 provided additional information confirming this
result. In particular, cleavage of the internal glycosidic bond
produced the sodium adduct of the digalactosyl ion at m/z 363.4
(C2 – 2H) and a sodium adduct ion at m/z 347.4 (B2) arising
from the loss of the hydroxyl group linked to the 1′ position of
the 6-O-substituted galactose (Figure 4). Additionally, a sodium

10294 J. Agric. Food Chem., Vol. 55, No. 25, 2007
Napolitano et al.
Figure 3. Positive HPLC-ESI/MS/MS spectrum and fragmentation pathway of the [M + Na]+ ion at m/z 829.9 derived from MGDG compound 11
showing the total spectrum (a), the high-mass region (b), and the low-mass region (c).
adduct ion at m/z 405.5 (E) was generated by the simultaneous
cleavage of the two neutral fatty acids, while further sodium
adduct ions at m/z 463.5 (2D1,2), 477.5 (3D1,2), m/z 533.7 ([M
+ Na – 162 – R1CO2H]+) and 551.7 ([M + Na – 162 –
R2CO2H]+) originated from the fragmentation pathway depicted
in Figure 4. Finally, the MS/MS spectrum also displayed ions
at m/z 293.3, 335.5, and 367.1 corresponding respectively to
the fragment ions [R1CO]+, [R2CO + 74]+, and [R1CO + 74]+.
The MS/MS spectra obtained for the galactolipids belonging
to the lower series of MGDG and DGDG compounds, i.e., those
characterized by C16:0, C18:0, C18:1, C18:2, C18:3, and C19:2
fatty acids linked to the glycerol backbone, were less informative
than those recorded for the higher series. Nevertheless, com-
parison of the mass spectra of the higher galactolipids with those
belonging to the lower series permitted the structural charac-
terization of the latter. According to this methodology, and
applying the two elution methods (A and B) in order to improve
the separation of galactolipids with different polarities, it was
possible to identify all galactolipids present in the analyte
mixture in terms of fatty acid composition, regiochemistry, and
position of double bonds on the respective acyl chains (Tables
1 and 2). The pure isolated galactolipids were quantified by
HPLC-ESI/MS, and the results obtained are reported in Tables
1 and 2. From these data it was estimated that MGDG
compounds comprised approximately 70% of the total amount
of galactolipids present in the nonpolar fraction.
DISCUSSION
The present study has demonstrated that galactolipids are
accumulated in the leaves of sweet potato, and 16 novel

Novel Galactolipids from the Leaves of I. batatas L.
J. Agric. Food Chem., Vol. 55, No. 25, 2007 10295
Figure 4. Positive HPLC-ESI/MS/MS spectrum and fragmentation pathway of the [M + Na]+ ion at m/z 991.9 derived from DGDG compound 24
showing the total spectrum (a), the high-mass region (b), and the low-mass region (c).
galactolipids (1, 6–14, 17, 22–26) together with 10 known
galactolipids were unambiguously identified (Tables 1 and 2)
by detailed spectrometric analysis.
should be defined as an 18:3 plant on the basis of the fatty acid
composition of the MGDG and DGDG spoecies present in the
leaves.
The abundance of galactolipid compounds characterized by
high contents of R-linolenic (C18:3 n-3, 6, 9 cis) and linoleic
(C18:2 n-6, 9 cis) acids explains, to some extent, the potential
interest in I. batatas as a nutraceutical plant. R-Linolenic and
linoleic acids are essential fatty acids that serve as precursors
for all n-3 and n-6 fatty acids, respectively. The essential nature
of R-linolenic acid has been well demonstrated (23, 24), and it
is known that deficiency leads to anomalies in the composition
of nerve membranes (25, 26) and in their architecture and
function (27, 28). Also noteworthy is the presence (confirmed
by GC-MS analysis) in sweet potato of the fatty acids C21:0
and C19:2, since these are rarely reported as constituents of
plant lipids.
Two main classes of MGDG compounds are found in higher
plants (22). In the first (and major) class, C18:3 fatty acids are
located at both the sn-1 and sn-2 positions of the glycerol
backbone: such C18:3/C18:3 galactolipids are found in all
eukaryotic lipids. In the second class, C18:3 fatty acids are
located at the sn-1 position, and a C16:3 fatty acid is present
exclusively at the sn-2 position of the glycerol: these C18:3/
C16:3 galactolipids are similar in structure to the glycerolipids
of the cyanobacteria and are referred to as prokaryotic type.
Almost all plants studied so far contain MGDG compounds with
the eukaryotic (C18:3/C18:3) structure, and some plants, such
as pea or cucumber, contain only eukaryotic MGDG compounds
and are known as “18:3 plants”. On the other hand, species of
several genera including Spinacia and Arabidopsis also contain
prokaryotic (C18:3/C16:3) MGDG compounds and are known
as “16:3 plants” (22). Following this classification, I. batatas
The successful isolation and characterization of the galacto-
lipid classes in leaves of I. batatas was achieved in the present
study by developing an adapted extractive method and an

10296 J. Agric. Food Chem., Vol. 55, No. 25, 2007
Napolitano et al.
LITERATURE CITED
analytical strategy based on mass spectrometry and NMR.
Several mass spectrometric approaches have been applied in
the determination of the structures of the fatty acyl groups in
glycerolipids. Thus, Orgambide et al. (29) employed metha-
nolysis of glycerolipid mixtures, followed by GC-MS analysis
of the fatty acid methyl ester derivatives, to determine fatty acid
composition in Rhizobium leguminosarum. An alternative
strategy used the collision-induced dissociation (CID) spectral
pattern of the carboxylate ion (RCOO-) obtained by negative
ion fast atom bombardment mass spectrometry (FAB/MS) of
glycerolipids in order to determine the structures of the fatty
acids (30, 31). However, using these methods it was not possible
to assign the detected fatty acid methyl ester derivatives or the
free carboxylate ions to the associated galactolipids present in
the mixture or to ascertain their positions on the glycerol
backbone. In contrast, Kim et al. (,32) were able to characterize
the structures of the MGDG and DGDG species in wheat flour
from the positive ion CID spectra of the sodium adduct ions
[M + Na]+ of galactolipids desorbed by FAB ionization.
Although it was possible to define the positions of the double
bonds on the acyl chains using this technique, the experiment
was carried out on pure compounds and thus does not represent
a reliable method for the analysis of mixtures. Moreover, while
electrospray ionization–quadrupole ion trap tandem mass spec-
trometry coupled with HPLC has been applied to the regio-
chemical determination of the acyl chains on the glycerol
backbone of MGDG and DGDG compounds, this approach is
not suitable to clarify the positions of the double bonds on the
acyl chains (20, 33).
(1) Villareal, R. L.; Tsou, S. C.; Lo, H. F.; Chiu, S. C. Sweetpotato
tips as vegetables. In Sweetpotato. Proceedings of the First
International Symposium; Villareal, R. L., Griggs, T. D., Eds.;
AVRDC: Shanhua, Taiwan 1982; pp 313–320.
(2) Islam, S. Sweetpotato (Ipomoea batatas L.) leaf: Its potential effect
on human health and nutrition. J. Food Sci. 2006, 71, 13–21.
(3) Islam, M. S.; Yoshimoto, M.; Terahara, N.; Yamakawa, O.
Anthocyanin compositions in sweetpotato (Ipomoea batatas L.)
leaves. Biosci., Biotechnol., Biochem. 2002, 66, 2483–2486.
(4) Islam, M. S.; Yoshimoto, M.; Yahara, S.; Okuno, S.; Ishiguro,
K.; Yamakawa, O. Identification and characterization of foliar
polyphenolic composition in sweetpotato (Ipomoea batatas L.)
genotypes. J. Agric. Food Chem. 2002, 50, 3718–3722.
(5) Osamu, M.; Itsu, N. Japanese Patent, JP7258100–1995-10-09, IPC:
A61K31/715; A61K31/715; (IPC1-7): A61K35/78 (+1). Appl No.
JP19940094047 19940324.
(6) Joyard, J.; Maréchal, E.; Block, M. A.; Douce, R. Plant galacto-
lipids and sulfolipids. Structure, distribution and biosynthesis. In
Membranes: Specialized Functions in Plants; Smallwood, M. ;
Knox, P.; Bowles, D. J., Eds.; BIOS Scientific Publishers: Oxford,
U.K., 1996; pp179–194.
(7) Reshef, V.; Mizrachi, E.; Maretzki, T.; Silberstein, C.; Loya, S.;
Hizi, A.; Carmeli, S. New acylated sulfoglycolipids and digalac-
tolipids and related known glycolipids from cyanobacteria with a
potential to inhibit the reverse transcriptase of HIV-1. J. Nat. Prod.
1997, 60, 1251–1260.
(8) Shirahashi, H.; Murakami, N.; Watanabe, N.; Nagatsu, A.;
Sakakibara, J.; Tokuda, H.; Nishino, H.; Iwashima, A. Isolation
and identification of anti-tumor-promoting principles from the
fresh-water cyanobacterium Phormidium tenue. Chem. Pharm.
Bull. 1993, 41, 1664–1666.
In the present study, a combination of two different mass
spectrometric techniques permitted the determination of the fatty
acid composition of each galactolipid compound by providing
information about acyl chain length, regiochemical distribution,
and positions of the double bonds. In this way, all of the
structural features of the galactolipids present in the leaves of
I. batatas were elucidated. The present study describes the first
application in this field of ESI/Q-TOF/MS combined with
HPLC.
(9) Shirahashi, H.; Marimoto, T.; Nagatsu, A.; Murakami, N.; Tatta,
K.; Sakakibara, J.; Tokuda, H.; Nishino, H. Antitumor-promoting
activities of various synthetic 1-O-acyl-3-O-(6′-O-acyl-ꢀ-D-ga-
lactopyranosyl)-sn-glycerols related to a natural product from
freshwater cyanobacterium Anabaena flos-aquae f. flos-aquae.
Chem. Pharm. Bull. 1996, 44, 1404–1406.
(10) Morimoto, T.; Nagatsu, A.; Murakami, N.; Sakakibara, J.; Tokuda,
H.; Nishino, H.; Iwashima, A. Anti-tumour-promoting glycero-
glycolipids from the green alga, Chlorella Vulgaris. Phytochem-
istry 1995, 40, 1433–1437.
As already discussed, galactolipids differing only in the length
of their acyl chains showed two different types of MS/MS
fragmentation patterns. In order to explain this unusual behavior,
a preferential conformational state for the higher series of
galactolipids is postulated, perhaps involving a relaxed form of
the acyl chains that renders them more prone to fragmentation
than their shorter counterparts. The apparent complexity of the
high mass region in the relative MS/MS spectra of these galacto-
lipids can, in fact, be simply resolved, allowing the positions of
the double bonds on the acyl chains to be determined. These
results, in turn, permitted the full characterization of the lower
galactolipid series by comparison of their fragmentation patterns
with those obtained for the higher series.
(11) Murakami, A.; Nakamura, Y.; Koshimizu, K.; Ohigashi, H.
Glyceroglycolipids from Citrus hystrix, a traditional herb in
Thailand, potently inhibit the tumour-promoting activity of 12-
O-tetradecanoylphorbol 13-acetate in mouse skin. J. Agric. Food
Chem. 1995, 43, 2779–2783.
(12) Tokuda, H.; Nishino, H.; Shirahashi, H.; Murakami, N.; Nagatsu,
A.; Sakakibara, J. Inhibition of 12-O-tetradecanoylphorbol-13-
acetate promoted mouse skin papilloma by digalactosyl diacylg-
lycerols from the fresh water cyanobacterium Phormidium tenue.
Cancer Lett. 1996, 104, 91–95.
(13) Murakami, C.; Kumagai, T.; Hada, T.; Kanekazu, U.; Nakazawa,
S.; Kamisuki, S.; Maeda, N.; Xu, X.; Yoshida, H.; Sugawara, F.;
Sakaguchi, K.; Mizushina, Y. Effects of glycolipids from spinach
on mammalian DNA polymerases. Biochem. Pharmacol. 2003,
65, 259–267.
(14) Larsen, E.; Kharazami, A.; Christensen, L. P.; Christensen, S. B.
An antiinflammatory galactolipid from rose hip (Rosa canina) that
inhibits chemotaxis of human peripheral blood neutrophils in vitro.
J. Nat. Prod. 2003, 66, 994–995.
(15) Jayaprakasam, B.; Zhang, Y.; Nair, M. G. Tumor cell proliferation
and cyclooxygenase enzyme inhibitory compounds in Amaranthus
tricolor. J. Agric. Food Chem. 2004, 52, 6939–6943.
(16) Schmidt-Schultz, T.; Althaus, H. H. Monogalactosyl diglyceride,
a marker for myelination, activates oligodendroglial protein kinase
C. J. Neurochem. 1994, 62, 1578–1585.
(17) Matsufuji, M.; Nagamatsu, Y.; Yoshimoto, A. Effects of bacterial
glyceroglycolipid M874B on growth and TPA-induced differentia-
ABBREVIATIONS USED
1-D, one dimensional; CD3OD, deuterated methanol; DGDG,
digalactosyldiacylglycerol; GC-MS, gas chromatography–mass
spectrometry; HMBC, heteronuclear multiple bond correlation;
HPLC-ESI/Q-TOF/MS/MS, high-performance liquid chroma-
tography coupled with electrospray ionization–quadrupole time-
of-flight tandem mass spectrometry; HSQC, heteronuclear single-
quantum correlation; MGDG, monogalactosyldiacylglycerol;
NMR, nuclear magnetic resonance; TOCSY, total correlated
spectroscopy; UV, ultraviolet.

Novel Galactolipids from the Leaves of I. batatas L.
J. Agric. Food Chem., Vol. 55, No. 25, 2007 10297
tion of HL60 cells. Biosci., Biotechnol., Biochem. 2000, 64, 1302–
1304.
(18) Matsufuji, M.; Nagamatsu, Y.; Yoshimoto, A. Protective effects
of bacterial glyceroglycolipid M874B against cell death caused
by exposure to heat and hydrogen peroxide. J. Biosci. Bioeng.
2000, 89, 345–349.
(19) Sassaki, G. L.; Machado, M. J.; Tischer, C. A.; Gorin, P. A. J.;
Iacomini, M. Glycosyldiacylglycerolipids from the lichen Dicty-
onema glabratum. J. Nat. Prod. 1999, 62, 844–847.
(20) Guella, G.; Frassanito, R.; Mancini, I. A new solution for an old
problem: the regiochemical distribution of the acyl chains in
galactolipids can be established by electrospray ionization tandem
mass spectrometry. Rapid Commun. Mass Spectrom. 2003, 17,
1982–1994.
(21) Kim, Y. H.; Gil, J. H.; Hong, J.; Yoo, J. S. Tandem mass
spectrometric analysis of fatty acyl groups of galactolipid mo-
lecular species from wheat flour. Microchem. J. 2001, 68, 143–
155.
response study with long-chain n-3 fatty acids. Lipids 1992, 27,
98–103.
(27) Foote, K. D.; Hrboticky, N.; MacKinnon, M. J.; Innis, S. M. Brain
synaptosomal, liver, plasma, and red blood cell lipids in piglets
fed exclusively on a vegetable-oil-containing formula with and
without fish-oil supplements. Am. J. Clin. Nutr. 1990, 51, 1001–
1006.
(28) Zérouga, M.; Beaugé, F.; Niel, E.; Durand, G.; Bourre, J. M.
Interactive effects of dietary (n-3) polyunsaturated fatty acids and
chronic ethanol intoxication on synaptic membrane lipid composi-
tion and fluidity in rats. Biochim. Biophys. Acta 1991, 1086, 295–
304.
(29) Orgambide, G. G.; Huang, Z.-H.; Gage, D. A.; Dazzo, F. B.
Phospholipid and fatty acid compositions of Rhizobium legumi-
nosarum biovar trifolii ANU843 in relation to flavone-activated
pSym nod gene expression. Lipids 1993, 28, 975–979.
(30) Jensen, N. J.; Tomer, K. B.; Gross, M. L. Fast atom bombardment
and tandem mass spectrometry of phosphatidylserine and phos-
phatidylcholine. Lipids 1986, 21, 580–588.
(31) Jensen, N. J.; Tomer, K. B.; Gross, M. L. FAB MS/MS for
phosphatidylinositol, -glycerol, -ethanolamine and other complex
phospholipids. Lipids 1987, 22, 480–489.
(22) Heinz, E. Enzymatic reactions in galactolipid biosynthesis. In
Lipids and Lipid Polymers; Tevini, M., Lichtenthaler, H. K., Eds.;
Springer-Verlag: Berlin, Germany, 1977; pp 102–120.
(23) Lands, W. E. Renewed questions about polyunsaturated fatty acids.
Nutr. ReV. 1986, 44, 189–195.
(24) Simopoulos, A. P. Summary of the conference on the health effects
of polyunsaturated fatty acids in seafoods. J. Nutr. 1986, 116,
2350–2354.
(25) Connor, W. E.; Neuringer, M.; Lin, D. S. Dietary effects on brain
fatty acid composition: the reversibility of n-3 fatty acid deficiency
and turnover of docosahexaenoic acid in the brain, erythrocytes,
and plasma of rhesus monkeys. J. Lipid Res. 1990, 31, 237–247.
(26) Wainwright, P. E.; Huang, Y. S.; Bulman-Fleming, B.; Dalby,
D.; Mills, D. E.; Redden, P.; McCutcheon, D. The effects of
dietary n-3/n-6 ratio on brain development in the mouse: a dose
(32) Kim, Y. H.; Yoo, J. S.; Kim, M. S. Structural characterization of
sulfoquinovosyl, monogalactosyl and digalactosyl diacylglycerols
by FAB-CID-MS/MS. J. Mass Spectrom. 1997, 32, 968–977.
(33) Wang, W.; Liu, Z.; Ma, L.; Hao, C.; Liu, S.; Voinov, V. G.;
Kalinovskaya, N. I. Electrospray ionization multiple-stage tandem
mass spectrometric analysis of diglycosyldiacylglycerol glycolipids
from the bacteria Bacillus pumilus. Rapid Commun. Mass Spec-
trom. 1999, 13, 1189–1196.
Received for review May 7, 2007. Revised manuscript received October
2, 2007. Accepted October 4, 2007.
JF071331Z