Food Chemistry 158 (2014) 229238

Contents lists available at ScienceDirect

Food Chemistry
journal homepage: www.elsevier.com/locate/foodchem

Efcient preparative isolation and identication of walnut bioactive

components using high-speed counter-current chromatography and
LC-ESI-IT-TOF-MS
Mary H. Grace, Charles W. Warlick, Scott A. Neff, Mary Ann Lila
Plants for Human Health Institute, Food Bioprocessing and Nutritional Sciences, North Carolina State University, North Carolina Research Campus, Kannapolis, NC 28081, USA

a r t i c l e

i n f o

Article history:
Received 11 December 2013
Received in revised form 7 February 2014
Accepted 19 February 2014
Available online 1 March 2014
Keywords:
Walnut
Juglans regia
Ellagitannins
High-speed counter-current
chromatography
ESI-MS/MS

a b s t r a c t
Preparative isolation of complex mixtures of compounds from walnut polar extracts was established by a
combination of high-speed counter-current chromatography (HSCCC) and electrospray ionization-ion
trap-time of ight mass spectrometry (ESI-IT-TOF-MS). Compounds were isolated after a solvent optimisation selection based on solute distribution in a biphasic solvent system. Isolation was achieved through
one or two successive HSCCC runs, and nal purication on Sephadex LH-20. Isolated compounds
included ellagitannins (111), gallic acid (12), dicarboxylic acid glucosides (1315), hydrojuglone glucoside (16), catechin (17), procyanidin B2 (18), and megasterone glucosides (1920). Praecoxin D (4) was
isolated for the rst time from walnut, while praecoxin A methyl ester (5) and glansreginin A n-butyl
ester (14) are newly identied compounds. The purity and identity of isolated compounds were conrmed by NMR and HPLC-ESI-MS/MS. These results provided a foundation for in depth characterisation
of walnut compounds and offered an efcient strategy for isolation of potentially health-relevant phytochemicals from walnuts.
2014 Elsevier Ltd. All rights reserved.

1. Introduction
Walnuts (Juglans regia L.) are an excellent source of essential
unsaturated fatty acids (linoleic and a-linolenic acids), tocopherols, and the strong antioxidant hormone, melatonin (Amaral,
Casal, Pereira, Seabra, & Oliveira, 2003; Kornsteiner, Wagner, &
Elmadfa, 2006). Among tree nuts, walnut is one of the two highest
ranked nuts for antioxidant capacity and total phenolic content
(Wu et al., 2004). Walnut seed is rich in a variety of polyphenolic
components, which contribute to the total antioxidant activity of
this nut (Li et al., 2006). Ellagitannins, also known as hydrolysable
tannins, characterise and dominate the intrinsic phenolic prole of
walnuts (Li et al., 2006). In addition, the walnut is a resource for
quinones, phenyl propanoids (Colaric, Veberic, Solar, Hudina, &
Stampar, 2005), and dicarboxylic acid derivatives (Ito, Okuda,
Fukuda, Hatano, & Yoshida, 2007).
Consumption of walnuts, with their unique and diverse phytochemical composition, has been linked to inhibition of arteriosclerosis, hypercholesterolaemia and cardiovascular disease (Banel &
Corresponding author. Address: Plants for Human Health Institute, North
Carolina State University, North Carolina Research Campus, 600 Laureate Way,
Kannapolis, NC 28081, USA. Tel.: +1 704 250 5407; fax: +1 704 250 5409.
E-mail address: mlila@ncsu.edu (M.A. Lila).
http://dx.doi.org/10.1016/j.foodchem.2014.02.117
0308-8146/ 2014 Elsevier Ltd. All rights reserved.

Hu, 2009), hypertriglyceridaemia (Zibaeenezhad, Shamsnia, &

Khorasani, 2005), diabetes mellitus (Pan, Sun, Manson, Willett, &
Hu, 2013), and cancer (Davis et al., 2012). Walnuts have also been
shown to provide neuroprotective benets, improving memory
and cognition (Carey, Poulose, & Shukitt-Hale, 2012). Walnut ellagitannins were found to suppress liver injury induced by carbon tetrachloride and D-galactosamine (Shimoda et al., 2008).
Before further pharmacological evaluation of the health-protective roles of specic walnut constituents can occur, development of
efcient methods for isolation and gentle purication of large volumes of material, without degrading structural integrity, is warranted. Isolation of the predominant phytochemical group,
hydrolysable tannins, using normal-phase preparative chromatography is not suitable because it strongly adsorbs to silica gel
(Mueller-Harvey, 2001). The separation of tannins on columns of
hydroxypropylated dextran gel (i.e., Sephadex LH-20) is induced
by the differential adsorption of each compound on the gel, rather
than by gel ltration, and tannins of high molecular weight are
often difcult to recover from the column (Arapitsas, Menichetti,
Vincieri, & Romani, 2007). Better results have sometimes been
obtained using vinyl polymer gels such as Toyopearl HW-40 and
Diaion HP-20 (Mueller-Harvey, 2001), but the process is laborious
and time consuming if large quantities must be isolated.
High-speed counter-current chromatography (HSCCC) is a form of

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M.H. Grace et al. / Food Chemistry 158 (2014) 229238

liquidliquid partition chromatography which was rst invented

by Ito (1981). Solute separation is based on partitioning between
the two immiscible liquid phases: the mobile phase and the support-free liquid stationary phase. Without any solid matrix, the stationary phase is retained in the column with the aid of a centrifugal
force eld, so it eliminates irreversible adsorption of samples onto
the solid support. Therefore, HSCCC can provide a suitable alternative for large scale separation of polyphenols without degrading the
active compounds. Because of its high efciency, HSCCC has recently been used to effectively separate and purify a diversity of
natural products (Marston & Hostettmann, 2006), including a few
studies on the isolation of hydrolysable tannins (Engels, Ganzle, &
Schieber, 2010; Jikai, Yue, Henkel, & Weber, 2002; Liu, Su, Wang,
Gu, & Xing, 2010; Okuda et al., 1986). However, HSCCC has never
been used to isolate complex mixtures of compounds such as
walnut polyphenols.
In addition, electrospray ionization-ion trap-time of ight-mass
spectrometry (ESI-IT-TOF-MS) is a powerful technique for bioactive compound identication because it enhances high mass resolution and multiple fragmentations. A recent study utilizing LC-ion
trap-Orbitrap-MS led to tentative identication of 120 polyphenolic compounds in walnuts (Regueiro et al., 2014). We hypothesised
that HSCCC in combination with HPLCMS/MS, a strategy that we
have successfully applied for isolation and characterisation of several other plant components (Grace et al., 2012; Mbeunkui, Grace,
Yousef, & Lila, 2012), could be targeted and adapted to provide a
selective and efcient method for isolation and characterisation
of the otherwise intractable walnut phytoactive constituents.
The objective of this research was to establish a method for rapid and effective separation of key walnut phytochemical components which preserves structural integrity and yields high
quantities and purities. This paper reports the successful preparative isolation of several walnut components using HSCCC. The
compounds were identied and structurally characterised using
comprehensive NMR and LC-ESI-IT-TOF MSn analytical protocols.
2. Materials and methods
2.1. Materials
California walnuts (J. regia L.) were obtained from the California
Walnut Commission and kept frozen at 80 C until extraction.
n-Hexane, tert-butyl methyl ether (TBME), acetonitrile, n-butanol,
n-propanol, ethyl acetate, methanol (HPLC grade), formic acid
and acetic acid (ACS reagent grade) were purchased from Fisher
Scientic (Waltham, MA). LCMS grade solvents were obtained
from Honeywell Burdick & Jackson (Muskegon, MI). Sephadex
LH-20, ellagic acid and gallic acid were obtained from Sigma
Aldrich (St. Louis, MO). Catechin and procyanidin B2 were
purchased from Chromadex (Irvine, CA).
2.2. Apparatus
Preparative HSCCC was performed on an Armen fully integrated
Centrifugal Partition Chromatography (CPC) spot instrument SCPC
2  500 (Armen Instrument, St.-Ave, France). This instrument is a
fully automated system consisting of a 2 CPC column compartment
(2  500 mL), a pump, an injector, a UV/Vis detector, a fraction
collector, a 30-mL sample loop, panel PC with digital touch
screen, and Armen Glider CPC software (Armen Instruments). The
revolution speed of the rotors can be regulated with a speed
controller in the range between 0 and 20,000 rpm. HPLC was performed on an integrated Agilent 1200 series Rapid Resolution LC
System using a Phenomenex Synergi 4 lm Hydro-RP 80A column
(250 mm  4.6 mm  5 lm; Phenomenex, Torrance, CA). The
mobile phase consisted of 2% acetic acid in distilled H2O (solvent A)

and 0.5% acetic acid in 50% acetonitrile in water (solvent B).

The ow rate was set at 1 mL/min with a step gradient: 1055%
B over 10 min, 55100% B for 3 min, 10010% for 2 min, isocratic
at 10% for 5 min. LCMS analysis was performed using a Shimadzu
LCMS-IT-TOF instrument (Shimadzu, Tokyo, Japan) equipped with
a Prominence HPLC system (SIL-20A HT autosampler, LC-20AD
pump system, SDP-M20A photo diode array detector). The LC separation was performed using a C18 reverse-phase column (Shimpack XR-ODS column, 50 mm  3.0 mm id  2.2 lm; Shimadzu
Scientic Instruments Inc., Columbia, MD) with a binary solvent
system comprising of 0.1% formic acid in H2O (A) and 0.1% formic
acid in methanol (B). Compounds were eluted into the ion source
at a ow rate of 0.35 ml/min with a step gradient of 595% B over
30 min, isocratic at 95% B over 2 min, and return to 5% B over
1 min. Prior to the next injection, the column was re-equilibrated
for 5 min at initial conditions (5% B). The heat block and curved
desolvation line (CDL) were maintained at 200 C. Nitrogen gas
was used as nebuliser and drying gas with the ow rate set at
1.5 L/min. The ESI source voltage was set at 4.5 kV and the detector
was set at 1.5 V. The instrument was calibrated to <5 ppm error in
mass accuracy with an external standard of sodium TFA solution.
Ionization was performed using a conventional ESI source in
negative ionization mode. Data was acquired from m/z 150
1500. Shimadzus LCMS Solution software (Shimadzu Scientic
Instruments Inc.) was used for system control and data analysis.
NMR spectra were recorded on a Bruker Avance 700 MHz spectrometer (Bruker BioSpin Corporation, Billerica, MA), located at
DHMRI, NCRC, Kannapolis, NC.
2.3. Preparation of the crude extracts
Ground walnut kernels were defatted with n-hexane (1:2 w/v,
6); hexane was evaporated to afford the walnut oil. The defatted
powdered material was extracted with 80% methanol (1:2 w/v,
3), and the combined aqueous methanol extract was evaporated,
using a rotary evaporator, to an aqueous fraction. This aqueous
fraction was partitioned with ethyl acetate (1:3 v/v, 5), solvent
evaporated and the residue was freeze-dried to afford the ethyl
acetate fraction (EtOAc). The aqueous layer was subsequently partitioned with n-butanol (1:3 v/v, 3), solvent evaporated and the
residue was freeze-dried to afford the butanol fraction (BuOH).
The extraction and fractionation scheme is detailed in Fig. 1.
2.4. Solvent selection and HSCCC separation
The chromatographic process in HSCCC is based on the partition
of a solute between the two liquids that are used as the mobile and
stationary phases, respectively. Successful separation requires a
suitable choice of the two-phase solvent system, which provides
an ideal distribution ratio/partition coefcient (kD). The selection
of the most efcient two-phase solvent system for the target compound(s) is the most important step in the HSCCC process, and may
consume up to 90% of the time in procedure development.
A series of experiments were performed to determine the
kD-value in different solvent systems as determined by HPLC.
Approximately 2 mg of each extract or fraction were weighed in
a 15-mL centrifuge tube, to which 2 mL of each phase of the
pre-equilibrated two-phase solvent system were added. After the
tube was shaken vigorously, the solution quickly separated; then
the upper and the lower phases were analyzed by HPLC to obtain
the distribution coefcient of the target compound(s). The kD-value
was expressed as the peak area of the target compound(s) in the
upper phase divided by that of the lower phase. A set of solvent
systems were tested in order to optimise fractionation of each
extract, based on the calculated kD-values (Ito, 2005; Okuda
et al., 1986).

M.H. Grace et al. / Food Chemistry 158 (2014) 229238

231

Fig. 1. Overall work ow for isolation of walnut components using HSCCC. CPC 1: n-hexane:ethyl acetate:methanol:water (HEMWat) 0.25:5:0.25:5; CPC 2: n-butanol:npropanol:water 2:1:3; CPC 3: tert-butyl methyl ether:acetonitrile:water 3:1.5:4:5; CPC 4: n-butanol:methanol:water 2.5:0.5:5, CPC 5: n-butanol:ethyl acetate:water
0.5:2:2.5; CC: column chromatography on Sephadex.

For the HSCCC separation, the selected mixture of solvents were

shaken vigorously in a separatory funnel and equilibrated at room
temperature for 1 h. After each layer was degassed by sonication
for 15 min, the heavier denser phase (lower layer) was used as
the mobile phase, and the lighter phase (upper layer) was used
as the stationary phase (descending technique). The CPC column(s)
was rst lled with the upper stationary phase at 100 mL/min with
the column(s) rotating at 500 rpm. The columns speed was then increased to 1800 rpm and the lower mobile phase was pumped into
the inlet of the column at a ow rate of 10 mL/min. Equilibration
was reached when only the mobile phase was eluted from the column while the displaced volume of the stationary phase remained
constant. After equilibration, the sample was injected through the
sample loop. Walnut crude extracts or fractions (up to 2.0-g
batches) were dissolved in 25 mL of a mixture of upper and lower
phases, and ltered before loading into the sample loop. The UV
detection was performed from the outlet of the column and the
fractions were collected in 15-mL glass tubes. The mobile phase
was pumped into the inlet of the column at 10 mL/min for
150 min, and the stationary phase at the same ow rate for another
150 min. Collected fractions were concentrated and dried after
combining similar fractions based on the UV chromatogram displayed on the recorder. Complex fractions from CPC runs, as indicated by HPLC, were subjected to a second CPC after a solvent
selection procedure, using the same parameters as indicated above.
Fractions with good purity were combined together. Compounds
that were obtained with a purity less than 85% were re-puried
on a Sephadex LH-20 column using a step gradient of 50%, 70%

and nally 100% ethanol. An overview of the separation process

is summarised in Fig. 1.
2.5. Fractionation of the ethyl acetate and butanol extracts
The CPC of the EtOAc extract (2-g batches) was conducted using
solvent system n-hexane:ethyl acetate:methanol:water (HEMWat)
0.25:5:0.25:5 v/v/v/v, collecting 176  15-mL fractions over 5 h.
Fractions were pooled together into 12 samples according to the
UV chromatogram recorded at 280 nm in the CPC monitor
(Fig. 2). Samples were subjected to HPLC and those with similar
proles were combined to afford 6 main fractions E1E6. The
procedure was repeated several times to scale up the isolation.
A solvent selection procedure was run to select the best biphasic
solvent system for subsequent separation. Fraction E1 was
subjected to a CPC run using n-butanol:n-propanol:water 2:1:3
to afford 12 fractions. Compounds 1 and 2 were obtained in a
semi-pure form in fraction 1 and 4, respectively. Fraction E2 was
run on the CPC using tert-butyl methyl ether:acetonitrile:water
3:1.5:4:5 v/v/v/v to afford compounds 3 and 16. The latter needed
further purication on a Sephadex LH-20 using 50% methanol.
Fraction E3 was separated on a second CPC run using tert-butyl
methyl ether:acetonitrile:water 3:1.5:4:5 to afford 9 in a pure
form, while compounds 12 and 4 needed further purication on
Sephadex columns. Compound 6 eluted in a pure form in fraction
E4. Fraction E5 afforded compounds 17 and 18 after separation
on a Sephadex column eluted with 70% methanol. Fraction E6
afforded compound 10 (Fig. 1).

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M.H. Grace et al. / Food Chemistry 158 (2014) 229238

Fig. 2. HSCCCUV chromatograms of ethyl acetate (EtOAc) and butanol (BuOH) extracts. Solvent systems: EtOAc extract, n-hexane:ethyl acetate:methanol:water (HEMWat)
0.25:5:0.25:5 v/v/v/v (kD-value 0.74); BuOH extract, n-butanol:n-propanol:water 213 v/v/v (kD-value 0.89); ow rate 10 mL/min over 5 h; detection 280 nm. E1E6 and
B1B7 represent the combined fractions.

The BuOH extract (2-g batches) was fractionated on the CPC

using solvent system n-butanol:n-propanol:water 2:1:3 v/v/v. Seven main fractions (B1B7) were obtained after combining individual CPC fractions (Fig. 2) based on HPLC analysis. Fraction B1
contained compound 13 as a major component, which was puried
by a second CPC procedure using n-butanol:methanol:water
2.5:0.5:5 v/v/v. The same fraction also afforded 1 as a minor component. Fraction B2 contained a mixture of 1 and 13 that were separated on the CPC using the same solvent system as B1. Fraction B3
contained mainly compound 2. Fraction B4 was subjected to a second CPC run using butanol:methanol:water 2.5:0.5:5 v/v/v, and
compounds were further puried on Sephadex columns (5070%
ethanol) to afford 5 as a major component, and 8. Fraction B5 afforded 7, 11 and a mixture of 19 and 20, after CPC run using n-butanol:ethylacetate:water 0.5:2:2.5 v/v/v, and further purication on
Sephadex columns. Fraction B6 afforded 15 and 11 after CPC run
using n-butanol:ethylacetate:water 0.5:2:2.5 v/v/v. Fraction B7
contained a complex mixture of several compounds including, 11
and 15. It was subjected to CPC and Sephadex purication, same
as Fraction B6, to afford 14 as a minor component. The isolated
compounds were identied based on a combination of LCMS,
NMR analyses, and with the aid of published data and available
reference standards.

3. Results and discussion

3.1. HSCCC separation of compounds
In HSCCC separation, a suitable two-phase solvent system is
critical for a successful isolation and separation. The samples need

to have a favourable distribution coefcient in the two-phase

solvent system. The distribution ratio/partition coefcient (kD) is
dened as the mass concentration ratio of the solute in the stationary phase versus those in the mobile phase. The optimal kD-value
should be within the range of 0.671.5; small kD-values usually
result in a poor peak resolution, while large kD-values tend to produce excessive sample broadening (Marston & Hostettmann,
2006).
Based on the calculated kD-value, the two-phase solvent system
n-hexane:ethyl acetate:methanol:water (0.25:5:0.25:5) was
selected to separate the EtOAc extract, and n-butanol:n-propanol:water (2:1:3) was selected to separate the BuOH extract. Combined fractions according to the CPC-UV chromatogram (280 nm)
were explored by HPLC and LCMS analysis to tentatively identify
compounds before further purication. Fractions containing complex mixtures were further separated on a second CPC run after
optimization of the solvent system, and nal purication, if
needed, was achieved on a Sephadex LH-20. A roadmap of the
whole process is illustrated in Fig. 1 and the HSCCC chromatograms of rst CPC runs for the EtOAc and BuOH fractions are shown
in Fig. 2.
HSCCC of the ethyl acetate extract led to the isolation of tellimagrandin II (6) (Wilkins & Bohm, 1976) and ellagic acid (10) in fraction E4 and E6, respectively. Fraction E5 contained a mixture of
catechin (17) and procyanidin B2 (18) as indicated from HPLC
and LCMS analyses in comparison with reference standards. The
rest of the fractions (E1E3), were subjected to a second CPC run
after a solvent selection procedure to optimize separation. Fraction
E1 afforded 2,3-hexahydrodiphenoyl-glucoside (2,3-HHDP-glucoside) (1) (Seikel & Hillis, 1970) and pedunculagin (2) (Okuda,
Yoshida, Ashida, & Yazaki, 1983); E2 afforded tellimagrandin I (3)

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M.H. Grace et al. / Food Chemistry 158 (2014) 229238

(Wilkins & Bohm, 1976) as major component, in addition to 1,4,8trihydroxynaphthalene-1-O-b-glucoside (16) (Son, 1995); E3 afforded casuarictin (9) (Okuda et al., 1983), praecoxin D (4) (Hatano,
Yazaki, Okonogi, & Okuda, 1991), and gallic acid (12).
The BuOH extract afforded 7 main fractions; the largest weight
was concentrated in fraction B1 which showed glansreginin A (13)
(Ito et al., 2007) as a major component. Compound 13 was also obtained in a pure form with 2,3-HHDP glucoside (1) after a second
CPC run of fraction B2. Pedunculagin (2) was obtained in fraction
B3 in semi-pure form. Isostrictinin (8) (Okuda, Yoshida, Hatano,
Yazaki, & Ashida, 1980), and the new derivative praecoxin A
methyl ester (5) were obtained from fraction B4. Fraction B5 afforded strictinin (7) (Okuda et al., 1983), ellagic acid 4-O-xylopyranoside (11) (Tanaka, Jiang, & Kouno, 1998) and a 1:1 mixture of the
epimeric megastigmane compounds, blumenol C glucoside (19)
and byzantionoside (20) (Matsunami, Otsuka, & Takeda, 2010).
Fraction B6 afforded 2,7-dimethyl-2,4-diene-deca-a,x-diacid (16)
(Hegde et al., 2005), in addition to compound 11. The new dicarboxylic acid ester, glansreginin A butyl ester (14), was obtained
as a minor component of the complex mixture in B7. ESI-TOFMS, MS/MS and UV maxima of the isolated compounds are presented in Table 1, and chemical structures are illustrated in Fig. 3.
The use of centrifugal partition chromatography for isolation of
hydrolysable tannins was previously reported by Okuda (Okuda
et al., 1986) using normal-phase development (lower layer as stationary phase) with n-butanol:n-propanol:water 4:1:5 as the solvent system, for a run duration of about 25 h. Efcient
preparative isolation was achieved, and accurate discrimination
of compounds which had very similar molecular structures (e.g.,
pedunculagin versus tellimagrandin I, and casuarictin versus tellimagrandin II). These same compounds were separated in the present work over only a 5-h run (Fig. 1), using n-hexane:ethyl
acetate:methanol:water (0.25:5:0.25:5) in the descending mode.
HSCCC was previously used for the isolation of the gallotannin
1,2,3,4,6-penta-O-galloyl-glucose from Acer truncatum using nhexane:ethyl acetate: methanol:water (0.25:5:1:5 v/v/v/v) (Zhao,
Gao, Ma, Bai, & Zhang, 2007). In another study, HSCCC was successfully applied to the one-step separation of ellagic acid and corilagin
from the methanolic extract of Phyllanthus urinaria L in 3.5 h, using
n-butanol:acetic acid:water 4:1:5 (Jikai et al., 2002). HSCCC was recently applied to the one-step separation of geraniin, corilagin and

gallic acid, the main active constituents of Geranium wildfordii

Maxim, using n-hexane:ethyl acetate:methanol:acetic acid:water
(1:10:0.2:0.2:20 v/v/v/v) with > 91% purity (Liu et al., 2010). HSCCC
was also applied successfully to separate gallotannins from mango
kernels according to their degree of galloylation (tetra- to deca-Ogalloylglucose) using n-hexane:ethyl acetate:methanol:water
(0.5:5:1:5) over a 9-h run (Engels et al., 2010).
3.2. Identication and structure elucidation of isolated compounds
Ellagitannins, a group of polyphenolic compounds, dominated
the walnut EtOAc and BuOH extracts. In the negative ion mode
of ESI-MS, ellagitannins are shown as their deprotonated ions
[MH]. Eleven ellagitannins have been isolated from walnut extracts (111). MS/MS of these compounds indicated a common
fragment ion m/z 301 (Table 1), corresponding to ellagic acid (a
hydrolytic release of HHDP ester groups). This fragment ion is a
key feature of ellagitannins and considered to be a chemical marker compound for hydrolysable tannins (Seeram, Lee, Scheuller, &
Heber, 2006). Typical losses during fragmentation include galloyl
(151 amu), galloylglucose (331 amu), HHDP-glucose (481 amu)
and galloyl-HHDP-glucose (633 amu). MS/MS of 2, a bis-HHDPglucose molecule, showed a main fragment ion at m/z 481
[MHHDP]. Compound 3 showed a molecular ion peak m/z 785
with two extra protons compared to 2, and its MS/MS showed an
additional fragment ion at m/z 633 [M151] indicating the loss
of galloyl moieties rather than HHDP compared to 2. Compounds
9 and 6 showed [MH] at m/z 935 and 937, with MS/MS of m/z
783 (pedunculagin) and m/z 785 (tellimagrandin I), respectively,
due to the loss of galloyl moiety. Compounds 7 and 8 showed exactly the same molecular ion peak m/z 633 [MH] and same fragmentation pattern, but appeared at different retention times in the
LC (Table 1). The dicarboxylic acid compounds 13 and 14 shared a
fragment ion m/z 403, due to the loss of ester moiety attached at 600
of the glucose core. Compounds with an unsubstituted anomeric
proton were isolated as epimeric mixtures, and they appeared in
our HPLC and LCMS chromatograms as double peaks (see HPLC
of 13, 4 and 5, Fig. 4). Ellagitannins with gallic acid substituents
at position 1 of the glucose moiety (69) were isolated as single
compounds and appeared as a single peak in their HPLC (Fig. 4).
Praecoxin D (4), a known ellagitannin, was isolated for the rst

Table 1
Characterization of walnut compounds using HPLC-ESI-IT-TOF-MS in the negative ion mode.
#

tR (min)

Measured
m/z [M-H]-

Predicted
m/z [M-H]-

Error (ppm)

MS/MS (m/z)

kmax (nm)

Molecular
formula

Identication

1
2
3
4
5
6
7
8
9
10
11
12
13
14
15

1.62, 1.90
3.63, 6.60
5.04, 7.37
18.75, 23.16
13.30, 21.30
21.58
12.44
6.97
11.21
24.46
24.28
4.03
24.47
26.25
24.41

481.0622
783.0720
785.0866
933.0669
965.0900
937.0945
633.0764
633.0761
935.0972
300.9991
433.0418
169.0216
592.2042
648.2668
403.1608

481.0624
783.0686
785.0843
933.0640
965.0902
937.0953
633.0733
633.0733
935.0953
300.9990
433.0412
196.0213
592.2036
648.2662
403.1610

0.42
4.34
0.51
3.11
0.21
0.85
4.90
4.90
2.03
0.33
1.39
0.25
1.01
0.93
0.50

421,
481,
633,
785,
783,
785,
481,
481,
783,
301
403,
403,
223,

258
232
275, 222
280, 225
260,222
278, 237
267, 230
265, 221
277, 221
254
360, 253
280
266
266
265

C20H18O14
C34H24O22
C34H26O22
C41H26O26
C42H30O27
C41H30O26
C27H22O18
C27H22O18
C41H28O26
C14H6O8
C19H14O12
C7H6O5
C28H35NO13
C32H43NO13
C18H28O10

16

16.34

337.0916

337.0929

3.86

219

C16H18O8

17
18
19
20

10.61
11.40
25.00
25.00

289.0710
577.1378
371.2079
371.2079

289.0718
577.1352
371.2075
371.2075

2.77
4.50
1.08
1.08

245, 203, 161

289, 205
285
285

278
278
245
245

C15H14O6
C30H26O12
C19H32O7
C19H32O7

2,3-O-HHDP-D-glucoside
Pedunculagin
Tellimagrandin I
Praecoxin D
Praecoxin A methyl ester
Tellimagrandin II
Strictinin
Isostrictinin
Casuarictin
Ellagic acid
Ellagic acid 4-O-xyloside
Gallic acid
Glansreginin A
Glansreginin A butyl ester
2,7-Dimethyl-2,4-diene-deca-a,
x-diacid 8-O-b-glucoside
1,4,8-Trihydroxynaphthaline-1O-b-glucoside
(+)-Catechin
Procyanidin B2
Blumenol C glucoside
Byzantionoside B

tR, Retention time; #, compound numbers in Fig. 3.

301
301
481,
451,
481,
633,
301
301
633,

301
301
301
483, 301

481, 301

343, 283, 241

343, 297, 279, 241
161

234

M.H. Grace et al. / Food Chemistry 158 (2014) 229238

OH

HO

HO
O

OH

HO

HO

OH

HO

OHHO

O
O
O

C
O

HO

O
CO

CO

OH

CO O
CH2
O
O
OC
O
HO
OH
OC

HO
OH

HO
O

O
OC

CO
HO

OHHO

CH2

HO
HO

HO

10"

8"

6"

OH

11"

1"

2"

[12]

OH

12

10

HO
HO
HO

OH

O
OH

OH

OH

COOH

CH2

7 6

COOH

11

[15]

OR

OR

OH
OH

OH

OH

HO

OH

1'

OH

6'

OH

HO

3"

OH

[16]

R= H, [13]
R= n-Bu, [14]

OH

CO O

OH

OH

HO

4a

HO

5"

4"

H
N
2

OH

7"

8a

9"

12"

HO

OH

OH

OH HO

OR

OH

OH O

OH

[9]

O
C

OH

C
O
OH

HO

R = H, [ 10]
R = xylose, [11]

[8]

HO

O
CO OC

OR

OH

OH
HO

OH

OH

OC

OH

HO

OH

OH

HO
HO

CH2

O
O

HO

CH2

C
O
C
O

HO
HO

OH

[7]
HO

[5]

OH

[6]

HO

OH

OHHO

OH

OH

OH

OH HO

HO
OH
OH

HO

OH

C
O
OH

OH

O
CO

HO

HO

HO
O

CO

OH

[4]

OH

CH2

O
O

C
O

HO

OH

OHHO

OH

O
CO OC

OH

O
C

CH2

O
C
O O

HO

OH
HO

O
C O

HO
HO

OH

HO
HO

HO

[3]

OH

HO

OH

OH

HO

OH

HO

O
O

OH

HO

H3C OOC

CH2

OH

HO

OH

OH

HO
HO

HO
HO

OH

OH

O
C

O
CO OC

HO

[2]

CH2

O
O

C
O

OH

OH
O C
O

OH

HO

[1]

O
C O

HO
HO

O
CO

CO

OH

OH

CH2
O

O
O

C
O

HO

OH
OHHO

HO

O
C O

HO
HO

O
CO

CO

HO

OH

HO

CH2

OH
OH

[17]

[18]

R = glucose [19] R = glucose [20]

Fig. 3. Chemical structures of compounds isolated from walnut.

time from J. regia, while praecoxin A-methyl ester (5) was identied as a new ellagitannin derivative.
Praecoxin A methyl ester (5) was obtained as a light brown
powder. It showed a molecular ion at m/z 965 [MH] corresponding to the molecular formula C42H30O27, with extra 14 mass units
compared to praecoxin A. MS/MS of 5 resulted in fragment ions
at m/z 783 (pedunculagin), m/z 481 (HHDP-glucoside) and m/z
301 (ellagic acid). 1H NMR spectrum indicated a characteristic
ellagitannin skeleton with a HHDP group, a valoneoyl group, and
a glucose pyranose core as revealed by its close similarity to that

of praecoxin A (Hatano et al., 1991). Duplication of almost all signals in its NMR spectra indicated that 5 forms an epimeric mixture.
The characteristic signal at d 3.84 in the 1H NMR, calculated for
three protons, indicated that the carboxylic group of the valoneoyl
moiety had been methylated. This was also evident from the signal
at dC 53.02 in the 13C NMR spectrum.
Another group of compounds that was prominent in the BuOH
extract was the dicarboxylic acid glucopyranosides. Three
compounds could be isolated and identied; glansreginin A (13),
its butyl ester as a novel minor compound (14), and

M.H. Grace et al. / Food Chemistry 158 (2014) 229238

235

Fig. 4. HPLC chromatograms of ethyl acetate (EtOAc), butanol (BuOH) fractions and characteristic compounds isolated from walnut by HSCCC.

2,7-dimethyl-2,4-diene-deca-a,x-diacid 8-O-b-D-glucopyranoside
(15). Glansreginin A butyl ester (14) was obtained as a reddish
amorphous material. ESI-TOF MS showed molecular ion at m/z
648 [MH] corresponding to a molecular formula C32H43NO13.
The molecular weight of 14 exceeds that of 13 by 56 mass units, corresponding to an additional C4H8. MS/MS of 14 produced the same
fragment ions as 13, in addition to m/z 297 as the base peak (see
Table 1, and Fig. 5). This suggested that compound 14 has a very
close structure to that of 13 with the terminal carboxylic acid moiety
esteried with a butyl group. 1H NMR and 13C NMR were very close
to 13 with additional signals corresponding to a butyl ester. An additional methyl group appeared in 1H NMR at d 0.95 as a double
(J = 7.0 Hz), correlated to dC 13.0 in the HMQC spectra. The CH3
(CH2)2CH2 appeared as doublet of doublet at d 4.06, 3.82 in the

H NMR correlated to dC 64.7 in the HMQC spectra. The CH3CH2

CH2CH2 appeared as two multiplets at d 1.60 and 1.39 correlated
with a negative signal in the DEPT-135 spectra at dC 31.2. This was
the same as with the proton signals of CH3CH2(CH2)2CH2
located d 1.38 in the 1H NMR, which correlated with a negative DEPT
signal at dC 19.6. Altogether, the HMBC correlations proved that the
terminal carboxylic group has been esteried with an n-butyl
moiety.
Compound 15 was isolated for the rst time from walnut. Its
ESI-TOF MS indicated a molecular ion at m/z 403 [MH], corresponding to a molecular formula C18H28O10. The compound was
identied as 2,7-dimethyl-2,4-diene-deca-a,x-diacid 8-O-b- glucoside, based on comprehensive NMR analysis and comparison
with the published data for an antitumour agent identied in

236

M.H. Grace et al. / Food Chemistry 158 (2014) 229238

Fig. 5. ESI-MS and MS/MS of glansreginin A n-butyl ester (14) in the negative ion mode.

Oryctanthus sp (Hegde et al., 2005). A recent analysis tentatively

identied this compound as 8-hydroxy-2,7-dimethyl-2,4-decadiene-1,
10-dioic acid 6-O-b-glucoside based on MS/MS (Gomez-Caravaca,
Verardo, Segura-Carretero, Caboni, & Fernandez-Gutierrez, 2008).
Compound 15 has the same molecular formula and similar fragmentation pattern in MS/MS as the published compound; however,
we have determined that the structure of is 2,7-dimethyl-2,4diene-deca-a,x-diacid 8-O-b-glucoside, as illustrated in Fig. 3.
Compound 16 is a hydrojuglone derivative identied as 1,4,8trihydroxynaphthalene-1-O-b-glucoside by comparison of its spectral data with those for a compound isolated from the bark and
fruits of Manchurian walnut (Juglans mandshuric) (Son, 1995). This
compound and other structurally related compounds have been reported for their cytotoxicity (Liu, Satou, Koike, Li, & Nikaido, 2004),
inhibition of HIV-1 reverse transcription (Min, Nakamura,
Miyashiro, Kim, & Hattori, 2000), and strong inhibition of pancreatic lipase enzyme (Han et al., 2007).
Compounds 19 and 20 are two megastigmane isomers, which
have been recently identied in the seed coat of J. regia (Liu
et al., 2012). They were isolated as a 1:1 epimeric mixture, as
indicated by doubling of signals in their NMR spectra. They
appeared as one peak in our HPLC (Fig. 4). ESI-EI-TOF-MS gave
a molecular ion peak at m/z 417 [M+HCOOHH], in addition
to m/z 371 [MH]  corresponding to molecular formula
C19H32O7. The compounds were identied as blumenol C glucoside (19) and byzanthionoside B (20) based on their NMR data,
and comparison to those reported in the literature (Matsunami
et al., 2010).
Praecoxin A methyl ester (5); light brown powder. ESI-TOF-MS
m/z: 965.0900 [MH] (calculated m/z 965.0902), corresponding
to a molecular formula C42H30O27; UV kmax: 237 nm. 1H NMR
(700 MHz, in acetone-d6) d: 6.90 [a-anomer (a) and b-anomer

(b); 1H, s, valoneoyl (Val) H-600 ], 6.63 (a), 6.61 (b) (1H in total, each
s, Val H-30 ), 6.60, 6.59 (1H, each s, HHDP H-30 ), 6.58, 6.53 (1H, each
s, HHDP H-3), 6.35 (1H, s, Val H-3). Glucose protons d: 5.48 (d,
J = 4.5 Hz, H-1), 5.25 (dd, J = 4.5, 9.7 Hz, H-2), 5.26 (t, J = 9.7 Hz,
H-3), 4.86 (t, J = 9.0 Hz, H-4), 4.63 (dd, J = 6.7, 9.7 Hz, H-5), 5.22
(dd, J = 6.7, 13.5 Hz, H-6), 3.80 (d, J = 13.5 Hz, H-6) (a-anomer);
5.08 (d, J = 9.8 Hz, H-1), 5.10 (m, J = 4.5, 9.7 Hz, H-2), 5.12 (t,
J = 9.7 Hz, H-3), 4.86 (t, J = 9.0 Hz, H-4), 4.24 (dd, J = 6.7, 9.7 Hz,
H-5), 5.22 (dd, J = 6.7, 13.5 Hz, H-6), 3.87 (d, J = 12.8 Hz, H-6) (banomer); 3.84 (s, OCH3). 13C NMR (175 MHz, acetone-d6) dC:
53.02 (OCH3), 63.94 [glucose(glc) C-6, a and b], 67.5 (glc C5, a),
69.67 (glc, C-4, b), 70.02 (glc C-4, a), 72.38 (glc C-5, b), 75.72 (glc
C-2, a), 75.65 (glc C-3, a), 77.25 (glc C-3, b), 78.44 (glc C-2, b),
91.91 (glc C-1, a), 95.54 (glc C-1, b), 107.14, 107.24, 107.44,
107.51, 107.60, 107.79 ((Val C-3, HHDP C-3 and C-30 ), 108.34 (Val
C-600 ), 108.50 (Val C-30 ), 113.97, 114.00, 114.08 (Val C-100 , HHDP
C-10 ), 114.86, 114.93 (HHDP C-1), 115.77, 116.06, 116.07 (Val C1
and C10 ), 125.31, 125.35 (Val C-2), 126.57, 126.60, 126.69,
126.89, (HHDP C-2 and C 20 ), 131.72, 131.77 (Val C-20 ), 135.95,
136.03, 136.50, 136.68, 136.69, 136.84 (Val C-50 , C-5, HHDP C-5,
C-50 ), 139.59, 139.97, 140.46 (Val C-200 and C-300 ), 142.44 (Val C400 ), 144.28, 144.32, 144.37, 144.44 (Val C-500 , HHDP C-6 and C60 ), 144.7, 145.09, 145.14, 145.2, 145.39 (Val C-4, C-6, and C-60 ,
HHDP C-4 and C-40 ), 149.08, 149.12, 149.30,149.35 (Val C-40 ),
167.87, 167.90, 167.96 (Val C-70 and C-700 ), 168.79, 168.89 (Val C7), 169.28, 169.36, 169.45 (HHDP C-70 and C-7).
Glansreginin A-butyl ester (14); light reddish amorphous material. ESI-TOF-MS m/z: 648.2668 [MH] (calculated m/z 648.2662),
corresponding to a molecular formula C32H43NO13; UV kmax:
266 nm. 1H NMR (700 MHz, in acetone-d6) d: 7.45 (d, J = 7.4 Hz,
H-5), 7.28 (t, J = 7.4 Hz, H-7), 7.21 (d, J = 11.5 Hz, H-300 ), 7.02 (t,
J = 7.4 Hz, H-6), 6.90 (d, J = 7.4 Hz, H-8), 6.51 (dd, J = 11.5,

M.H. Grace et al. / Food Chemistry 158 (2014) 229238

14.4 Hz, H-400 ), 6.23 (dt, J = 14.5, 7.5 Hz, H-500 ), 4.04 (dt, J = 8, 4.5 Hz,
H-800 ), 3.30 (2H, m, H-3), 2.47, 2.1 (2H, dt, J = 14.5, 7.5 Hz, H-600 ),
2.45 (2H, m, H-900 ), 1.91 (s, CH3-1100 ), 0.93 (3H, CH3-1200 ). Glucose
protons: d 4.5 (d, J = 7.4 Hz, H-10 ), 3.20 (t, J = 9 Hz, H-20 ), 3.37 (t,
J = 9 Hz, H-30 ), 3.23 (t, J = 9.0 Hz, H-40 ), 3.26 (m, H-50 ), 4.14, 4.30
(2H, d, J = 5.5, 12.0 Hz, H-60 ). n-Bu protons: d 0.93 (3H, d,
J = 7.0 Hz, CH3), 1.37 (2H, m, CH3CH2), 1.60 and 1.39 (2H, m,
CH2CH2O), 4.06, 3.82 (2H, m CH3(CH2)2CH2O). 13C NMR
(175 MHz, acetone-d6) dC: 12.8 (C-1100 ), 14.0 (C-1200 ), 37.1 (C-600 ),
39.0 (C-700 ), 40.4 (C-3), 70.8 (C-800 ), 78.5 (C-4), 110.5 (C-8), 122.4
(C-6), 126.0 (C-200 and C-5), 127.9 (C-400 ), 128.0 (C-4a), 130.9 (C7), 139.3 (C-300 ), 142.8 (C-8 and C-500 ), 168.7 (C-100 ), 172.9 (C-8a),
176.8 (C-2 and C-1000 ). Glucose: dC 99.1 (C-10 ), 74.3 (C-20 ), 77.5
(C-30 ), 70.7 (C-40 ), 75.0 (C-50 ), 64.3 (C-60 ). n-Bu: dC 13.0 (CH3-),
19.6 (CH2), 31.2 (CH2), 64.7 (CH2O).
4. Conclusions
A rapid efcient method for preparative isolation of walnut polar components was applied utilizing a combination of HSCCC and
HPLCMS/MS techniques. Twenty compounds could be isolated in
one or two successive CPC runs and nal purication was accomplished on Sephadex LH-20. Compounds that have been isolated
were ellagitannins (111), gallic acid (12), dicarboxylic acid glucosides (1315), hydrojuglone glucoside (16), catechin (17), procyanidin B2 (18), and megastigmane glucosides (1920). Praecoxin
A-methyl ester is a new ellagitanin derivative which was abundant
in walnut BuOH extract. Another new compound, glansreginin A nbutyl ester, was isolated as a minor compound from the BuOH extract. The isolation and comprehensive NMR study enabled the
conrmation of the structure of 15 as 2,7-dimethyl-2,4-dienedeca-a,x-diacid 8-O-b-glucoside, which had previously been misidentied in another report. The approach was found to be more
efcient in terms of separation time, solvent cost, the number of
compounds recovered, and an ability to preserve the integrity of
compounds which would otherwise degrade upon contact with solid supports. The procedure we developed provided a foundation
for further development and exploration of more walnut compounds, and furnished the ability to perform large-scale isolation
of compounds that can be used for studies of health benecial
activities of walnut components.
Acknowledgements
This work was funded by a grant from the California Walnut
Commission. M.A.L. was the principle investigator on the grant.
M.G. designed the experiments, interpreted the results, and wrote
the manuscript. C.W. did the extraction and purication of compounds. S.N. helped with the NMR experiments.
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