Identification of the characteristic components in walnut and anti-inflammatory effect of glansreginin A as an indicator for quality evaluation

Article abstract of DOI:10.1080/09168451.2019.1670046

Walnut is a nutritious food material, but only a few studies have been conducted on the mechanisms of its functions and the technique for quality evaluation. Therefore, we analyzed the components in aqueous methanol extract of walnut, and characterized 30 components, including three new compounds, glansreginin C, ellagic acid 4-O-(3′-O-galloyl)-β-D-xyloside, and platycaryanin A methyl ester. We analyzed the extracts of other nuts using HPLC and clarified that a characteristic peak corresponding to glansreginin A was mainly observed in walnut. These results suggested that glansreginin A might be an indicator component of the quality of walnut. We then examined whether glansreginin A has neuroprotective effect, using lipopolysaccharide (LPS)-induced inflammatory model mice. The results revealed that oral administration of glansreginin A prevented LPS-induced abnormal behavior and LPS-induced hyper-activation of microglia in the hippocampus. These results suggested that glansreginin A has the ability to exert neuroprotective effect via anti-inflammation in the brain.

Full text of DOI:10.1080/09168451.2019.1670046

Bioscience, Biotechnology, and Biochemistry
ISSN: 0916-8451 (Print) 1347-6947 (Online) Journal homepage: https://www.tandfonline.com/loi/tbbb20
Identification of the characteristic components
in walnut and anti-inflammatory effect of
glansreginin A as an indicator for quality
evaluation
Rie Haramiishi, Satoshi Okuyama, Morio Yoshimura, Mitsunari Nakajima,
Yoshiko Furukawa, Hideyuki Ito & Yoshiaki Amakura
To cite this article: Rie Haramiishi, Satoshi Okuyama, Morio Yoshimura, Mitsunari Nakajima,
Yoshiko Furukawa, Hideyuki Ito & Yoshiaki Amakura (2019): Identification of the characteristic
components in walnut and anti-inflammatory effect of glansreginin A as an indicator for quality
evaluation, Bioscience, Biotechnology, and Biochemistry, DOI: 10.1080/09168451.2019.1670046
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BIOSCIENCE, BIOTECHNOLOGY, AND BIOCHEMISTRY
https://doi.org/10.1080/09168451.2019.1670046
Identification of the characteristic components in walnut and anti-
inflammatory effect of glansreginin A as an indicator for quality evaluation
Rie Haramiishia, Satoshi Okuyamab, Morio Yoshimuraa, Mitsunari Nakajimab, Yoshiko Furukawab,
Hideyuki Itoc and Yoshiaki Amakuraa
b
aDepartment of Pharmacognosy, College of Pharmaceutical Sciences, Matsuyama University, Matsuyama, Japan; Department of
c
Pharmaceutical Pharmacology, College of Pharmaceutical Sciences, Matsuyama University, Matsuyama, Japan; Faculty of Health and
Welfare Science, Department of Nutritional Science, Okayama Prefectural University, Okayama, Japan
ABSTRACT
ARTICLE HISTORY
Received 30 July 2019
Accepted 12 September 2019
Walnut is a nutritious food material, but only a few studies have been conducted on the
mechanisms of its functions and the technique for quality evaluation. Therefore, we analyzed
the components in aqueous methanol extract of walnut, and characterized 30 components,
including three new compounds, glansreginin C, ellagic acid 4-O-(3′-O-galloyl)-β-D-xyloside,
and platycaryanin A methyl ester. We analyzed the extracts of other nuts using HPLC and
clarified that a characteristic peak corresponding to glansreginin A was mainly observed in
walnut. These results suggested that glansreginin A might be an indicator component of the
quality of walnut. We then examined whether glansreginin A has neuroprotective effect,
using lipopolysaccharide (LPS)-induced inflammatory model mice. The results revealed that
oral administration of glansreginin A prevented LPS-induced abnormal behavior and LPS-
induced hyper-activation of microglia in the hippocampus. These results suggested that
glansreginin A has the ability to exert neuroprotective effect via anti-inflammation in the
brain.
KEYWORDS
Walnut; glansreginin C;
ellagic acid 4-O-(3′-O-
galloyl)-β-D-xyloside;
glansreginin A; anti-
inflammation
Walnut (the seed of Juglans species) is one of the most
popular and nutritious nuts, and people from all over the
world have been consuming it since ancient times. There
are several kinds of walnut, and its representative cultivar
is Juglans regia L. (Juglandaceae), which is mainly culti-
vated in the United States and China [1]. With respect to
the nutritional components of walnut, the following has
been reported: 1) the content of lipids, proteins and
carbohydrates is 68.8, 14.6, and 11.7 g per 100 g, respec-
tively, indicating that the nutritive components with the
highest content in walnut is lipids; 2) among lipids, the
content of linoleic acids (ω-6 fatty acids), α-linolenic acid
(ω-3 fatty acids), and monounsaturated oleic acid of
essential fatty acids are in high content; 3) the balance
of amino acid components is better than that of other
vegetable proteins; and 4) the vitamins, minerals, and
dietary fibers are present in moderate quantities in wal-
nut [2,3]. Besides nutritional components, walnut has
been reported to contain polyphenols such as gallic
acid, ellagic acid, and hydrolyzable tannins [4,5]. With
respect to the biological activities of walnut, it prevents
various diseases and improves risk factors associated
with cardiovascular diseases [6,7], coronary heart disease
[3], diabetes [8], liver damage [9], hyperlipidemia and
metabolic syndrome [10], inflammation [11], and
Alzheimer’s disease (AD) [12,13]; it also maintains
intestinal environment [14], and metabolism [15].
These above-mentioned studies have shown the possibi-
lity that the fatty acids and polyphenols might contribute
to the various biological activities of walnut.
Walnut is thus an excellent source of phytochem-
ical compounds, but a variation in the content of
these compounds might be a problem when used as
natural medicine and functional food. One of the
aims of the present study was to identify the char-
acteristic component(s) contained in walnuts, pro-
viding the data for quality evaluation of walnut
products. We successfully revealed that glansreginin
A might be an indicator component in walnut.
A large amount of data shows that eating a walnut-
rich meal might prevent and improve risk factor asso-
ciated with lifestyle diseases [6–11,14,15]. Recent finding
also indicated that walnut might maintain brain health
and function [12,13]. The second aim of the present
study was to investigate whether glansreginin A has
effect(s) on the brain.
Materials and methods
General
Optical rotations were measured on the JASCO P-1020
digital polarimeter (JASCO Corporation, Tokyo, Japan).
UV spectra were recorded using Shimadzu UVmini-
1240 (Shimadzu Corporation, Kyoto, Japan) and
CONTACT Yoshiaki Amakura
amakura@g.matsuyama-u.ac.jp
© 2019 Japan Society for Bioscience, Biotechnology, and Agrochemistry

2
R. HARAMIISHI ET AL.
JASCO V-530 (JASCO Corporation, Tokyo, Japan).
Electrospray ionization mass spectroscopy (ESI-MS)
and high-resolution (HR) ESI-MS were performed with
the micrOTOF-Q (Bruker Daltonics, Billerica, MA,
USA) mass spectrometer using acetonitrile or methanol
(MeOH) as the solvents. The NMR spectra were
recorded on the Bruker AVANCE500 instrument
(Bruker BioSpin, Billerica, MA, USA; 500 and 126 MHz
Materials
Walnut, almond, pistachio, pecan nut (from USA),
cashew nut (from India), hazelnut (from Turkey),
and macadamia nut (from Australia) (all raw materi-
als) for the phytochemical investigation were pur-
chased from LOHAS (Sapporo, Japan) in 2015.
Lipopolysaccharide (LPS) from Salmonella enterica
serotype typhimurium was obtained from Sigma-
Aldrich Co. LLC. (St. Louis, MO, USA). All other
reagents used were of special or analytical grade.
1
for H and 13C, respectively); chemical shifts are pre-
sented as δ (parts per million) values relative to those of
solvent [MeOH-d4 (δH 3.30; δC 49.0) and acetone-d6 (δH
2.04; δC 29.8)] on a tetramethylsilane scale. The standard
pulse sequences programed for the instrument
(AVANCE 500) were used for 2D measurements
(COSY, HSQC, and HMBC). JCH was set at 10 Hz for
HMBC analysis. Column chromatography was carried
out using Diaion HP-20 (Mitsubishi Chemical Co.,
Tokyo, Japan), Chromatorex ODS (Fuji Silysia
Chemical Ltd., Aichi, Japan), Sephadex LH-20 (GE
Healthcare, Little Chalfont, UK), Toyopearl HW-40F
(Tosoh, Tokyo, Japan), YMC GEL ODS (YMC Co.
Ltd., Kyoto, Japan), and Sep-Pak Plus tC18 cartridge
(Waters, Milford, MA, USA). The normal-phase HPLC
was conducted on a SHISEIDO SILICA SG80 (Shiseido,
Tokyo, Japan) column (150 mm × 2.0 mm i.d.) devel-
oped with n-hexane-MeOH-tetrahydrofuran-formic
acid (55:33:11:1) containing oxalic acid (450 mg/L)
(flow rate: 0.5 mL/min; detection wavelength: 280 nm).
The reversed-phase (RP) HPLC conditions were as fol-
lows. Condition 1: column, YMC-pack ODS-AQ (5 μm,
150 mm × 2.0 mm i.d.; YMC Co. Ltd.); mobile phase,
10 mM phosphate buffer–acetonitrile (7:3–9:1); column
temperature, 40°C; flow-rate, 0.2 mL/min; and detection
wavelength, 254 or 280 nm. Condition 2: column, YMC-
pack ODS-AQ (5 μm, 150 mm × 2.0 mm i.d.; YMC Co.
Ltd.); mobile phase, 10 mM phosphate buffer–acetoni-
trile (75:25); column temperature, 35°C; flow-rate,
0.2 mL/min; and detection wavelength, 250 nm.
Condition 3: column, L-column ODS (5 μm, 150 mm
× 2.1 mm i.d. Chemicals Evaluation and Research
Institute, Tokyo, Japan); mobile phase, 0.1% formic
acid in distilled water (solvent A) and 0.1% formic acid
in acetonitrile (solvent B) (0–30 min, 0–50% B in A; 30–
35 min, 50–85% B in A; 35–40 min, 85% B in A; 40–50
min, 85–90% B in A; 50–55 min, 90–100% B in A; 55–60
min, 100% B in A); injection volume, 2 μL; column
temperature, 40°C; flow-rate, 0.3 mL/min; detection
wavelength, 254 nm. Condition 4: column, L-column
ODS (5 μm, 150 mm × 2.1 mm i.d.); mobile phase, 5%
acetic acid (solvent A) and acetonitrile (solvent B) (0–30
min, 0–50% B in A; 30–35 min, 50–85% B in A; 35–40
min, 85% B in A; 40–50 min, 85–90% B in A; 50–55 min,
90–100% B in A; 55–60 min, 100% B in A); injection
volume, 2 μL; column temperature, 40°C; flow-rate, 0.3
mL/min; detection wavelength, 200–400 nm.
Extraction and isolation
The crushed kernels of walnut (4.5 kg) were homoge-
nized in 80% aqueous MeOH (20 L). After filtration,
the filtrate was evaporated in vacuo to obtain 80%
MeOH extract (199.8 g). A part of this extract (190 g)
was separated by column chromatography over
Diaion HP-20 with aqueous MeOH solvent
(0:100→10:90→20:80→30:70→40:60→50:50→100:0)
in a stepwise gradient mode. Next, obtained each fraction
was separated and purified by repeated column chroma-
tography, and the fractions showing similar HPLC
(Condition 1) patterns were combined and further pur-
ified by column chromatography. The 10% MeOH eluate
(4.0 g) was separated by YMC GEL ODS column chro-
matography with aqueous MeOH to obtain glansreginic
acid 8-O-β-D-glucoside (15, 93.2 mg). The 30% MeOH
eluate (5.0 g) was separated by Sephadex LH-20 and
YMC GEL ODS column chromatography with aqueous
MeOH to obtain ellagic acid (7, 1.8 mg), 5-hydroxytryp-
tamine (12, 9.5 mg), and casuarictin (24, 697.6 mg). The
40% MeOH eluate (5.0 g) was separated by Toyopearl
HW-40F, Sephadex LH-20, and YMC GEL ODS column
chromatography with aqueous MeOH to obtain com-
pound 3 (platycaryanin A methyl ester) (6.7 mg), glans-
reginin A (5, 526.6 mg), ellagic acid (7, 2.1 mg),
1,2,4,6-tetra-O-galloyl-β-D-glucose (18, 32.9 mg),
1,2,3,4,6-penta-O-galloyl-β-D-glucose (19, 3.8 mg), tell-
imagrandin II (23, 76.1 mg), casuarictin (24, 153.6 mg),
pterocarinin C (26, 12.0 mg), rugosin C (27, 22.4 mg),
and euprostin A (30, 14.0 mg). The 50% MeOH eluate
(4.0 g) was separated by YMC GEL ODS, Sephadex LH-
20, and Chromatorex ODS column chromatography and
Sep-Pak Plus tC18 cartridge with aqueous MeOH to
obtain compound 1 (glansreginin C) (32.3 mg), com-
pound 2 (ellagic acid 4-O-(3′-O-galloyl)-β-D-xyloside)
(6.0 mg), compound 3 (platycaryanin A methyl ester)
(12.0 mg), glansreginic acid (4, 9.6 mg), glansreginin
A (5, 866.6 mg), ellagic acid (7, 116.1 mg), mixture of
blumenol C glucoside (9) and byzantionoside B (10)
(22.8 mg), 1,2,3,4,6-penta-O-galloyl-β-D-glucose (19,
18.2 mg), and rugosin C methyl ester (28, 46.5 mg). The
MeOH eluate (2.0 g) was separated by Toyopearl HW-

CHARACTERISTIC COMPONENTS IN WALNUT
3
40F column chromatography with aqueous MeOH to
obtain valoneic acid dilactone methyl ester (13, 7.8 mg).
Additionally, the 80% aqueous MeOH extract
(6.6 g) obtained from a separate experiment using
walnut (100 g) in a similar manner was separated
by Diaion HP-20 column chromatography.
The 10–40% MeOH eluate was separated by YMC
GEL ODS, Sephadex LH-20, and Chromatorex
ODS column chromatography with aqueous
MeOH to obtain glansreginin A (5, 9.6 mg), methyl
gallate (6, 5.4 mg), (+)-catechin (8, 1.4 mg),
4′-dihydrophaseic acid β-glucopyranose ester (11,
2.5 mg), ellagic acid 4-O-β-D-xyloside (14,
10.3 mg), glansreginic acid 8-O-β-D-glucoside
(15, 1.5 mg) glansreginin B (16, 1.7 mg), 1,2,6-tri-
O-galloyl-β-D-glucose (17, 1.0 mg), 1,2,4,6-tetra-
O-galloyl-β-D-glucose (18, 1.9 mg), strictinin (20,
5.2 mg), isostrictinin (21, 3.9 mg), tellimagrandin
I (22, 5.6 mg), tellimagrandin II (23, 3.3 mg),
casuarictin (24, 15.9 mg), pedunculagin (25,
35.4 mg), rugosin C (27, 3.2 mg), and casuarinin
(29, 1.0 mg).
C44H52O21N2-H: 943.2990) and 967.2941 ([M+
Na]+, calculated for C44H52O21N2+ Na: 967.2955).
Compound 2 (Ellagic acid 4-O-(3′-O-galloyl)-β-
D-xyloside): A light brown amorphous powder. UV
λmax (MeOH) nm (log ε): 343 (4.03), 275 (4.54), 258
25
1
(4.53). [α]D + 35° (c = 0.1, MeOH). H-NMR (500
MHz, MeOH-d4) δ 7.74 (1H, s, H-5), 7.47 (1H, s,
H-5′), 7.16 (2H, s, galloyl H-2‴, 6‴), 5.21 (1H, t, J = 9
Hz, xyl H-3), 5.07 (1H, d, J = 7.5 Hz, xyl H-1), 4.10
(1H dd, J = 5, 10.5 Hz, xyl H-5), 3.89 (1H, ddd, J = 5,
9, 9.5 Hz, xyl H-4), 3.81 (1H, dd, J = 7.5, 9 Hz, xyl
H-2), 3.58 (1H, brt, J = 10.5 Hz, xyl H-5). 13C-NMR
(126 MHz, MeOH-d4) δ 168.2 (galloyl C-7‴), 161.6
(C-7), 161.4 (C-7′), 150.1 (C-4′), 149.0 (C-4), 146.4
(2C, galloyl C-3‴, 5‴), 145.4 (C-3), 141.4 (C-3′), 139.8
(galloyl C-4‴), 138.0 (2C, C-2, 2′), 121.7 (galloyl
C-1‴), 116.8 (C-1), 113.7, 113.6 (C-5, 1′), 111.7
(C-5′), 110.5 (2C, galloyl C-2‴, 6‴), 109.9 (C-6′),
107.3 (C-6), 104.3 (xyl C-1), 78.2 (xyl C-3), 73.0 (xyl
C-2), 69.4 (xyl C-4), 67.1 (xyl C-5). HR-ESI-MS m/z:
585.0525 ([M–H]−, calculated for C26H18O16-H:
585.0522) and 609.0465 ([M+ Na]+, calculated for
C26H18O16+ Na: 609.0487).
The known compounds were identified by direct
comparison with authentic specimens or by compar-
ing their spectral data with those reported in the
literature. The physical and spectral data of com-
pounds 1–3 are as follows.
Compound 3 (Platycaryanin A methyl ester):
A brown amorphous powder. UV λmax (MeOH) nm
24
(log ε): 280 (4.48), 222 (4.97). [α]D + 21° (c = 0.1,
MeOH). 1H-NMR (acetone-d6+ D2O, 9:1) δ 7.16 (2H,
s, galloyl-H), 6.87, 6.59, 6.57, 6.46, 6.39 (each
1H, HHDP and tergalloyl-H), 6.20 (1H, d, J = 8.5
Hz, glcH-1), 5.44 (1H, dd, J = 8.5, 10 Hz, glcH-3),
5.29 (1H, dd, J = 7, 13 Hz, glcH-6), 5.19 (1H, t, J = 10
Hz, glcH-4), 5.18 (1H, t, J = 8.5 Hz, glcH-2), 4.49
(1H, brdd, J = 7, 10 Hz, glcH-5), 3.89 (1H, d, J = 13
Hz, glcH-6), 3.82 (3H, s, -OCH3). 13C-NMR
(acetone-d6+ D2O) δ 169.5 (2C), 168.8, 168.2, 168.1
(HHDP C-7, 7′, tergalloyl C-7, 7′, 7″), 165.2 (galloyl
C-7), 149.3 (2C), 146.2 (2C), 145.2, 145.1 (2C), 144.9,
144.5, 144.4 (HHDP C-4, 4′, 6, 6′, tergalloyl
C-4, 4′, 6, 6′, galloyl C-3, 5), 142.2, 140.6, 140.0,
139.9, 139.7, 137.0, 136.8, 136.5, 136.2, 131.3
(HHDP C-5, 5′, tergalloyl C-2′, 2″, 3″, 4″, 5, 5′, 5″,
galloyl C-4), 126.1, 125.8, 124.9 (HHDP C-2, 2′, ter-
galloyl C-2), 119.5 (galloyl C-1), 116.5, 116.1, 115.0,
114.4, 113.9 (HHDP C-1,1′, tergalloyl C-1, 1′, 1″),
110.2 (2C) (galloyl C-2, 6), 108.1, 108.0, 107.6, 107.2
(2C) (HHDP C-3, 3′, tergalloyl C-3, 3′, 6″), 92.2 (glc
C-1), 77.1 (glc C-3), 76.0 (glc C-2), 73.3 (glc C-5),
69.1 (glc C-4), 63.4 (glc C-6), 52.9 (-OCH3). HR-ESI-
MS m/z: 1117.1002 ([M–H]−, calculated for C49H34
O31-H: 1117.1011).
Compound 1 (Glansreginin C): A brown amor-
phous powder. UV λmax (MeOH) nm (log ε): 264
23
(4.38), 213 (4.89). [α]D -143° (c = 0.1, MeOH).
1
The H-NMR (500 MHz, MeOH-d4) and 13C-NMR
(126 MHz, MeOH-d4) data are provided in Table 1.
HR-ESI-MS m/z: 943.2973 ([M–H]−, calculated for
Table 1. 1H- (500 MHz) and 13C-NMR (126 MHz) data of
compound 1 measured in MeOH-d4.
Position
δC
δH (J in Hz)
2, 2ʹ
3, 3ʹ
4, 4ʹ
4a, 4a’
5, 5ʹ
6, 6ʹ
7, 7ʹ
8, 8ʹ
8a, 8a’
9, 9’
glc 1, 1ʹ
2, 2ʹ
3, 3ʹ
4, 4ʹ
5, 5ʹ
6, 6’
179.9, 179.6
42.44, 42.38
79.5, 79.4
126.4, 126.1
126.8, 126.7
123.1 (2C)
131.7, 131.6
111.5, 111.4
145.0, 144.9
172.1 (2C)
99.8, 99.6
74.7, 74.6
77.74, 77.70
71.2, 71.1
75.5, 75.4
64.6, 64.3
170.1
3.17 (4H, m)
7.39 (2H, d, J = 8.0)
7.05, 7.03 (each 1H, t, J = 8.0)
7.31, 7.28 (each 1H, t, J = 8.0)
6.91, 6.86 (each 1H, d, J = 8.0)
4.23, 4.14 (each 1H, d, J = 8.0)
3.27 (2H, m)
3.16 (2H, m)
3.31, 3.19 (each 1H, m)
2.98, 2.92 (each 1H, m)
4.19, 4.09 (each 2H, m)
1″
2″
3″
4″
5″
6″
7″
126.1
140.4
128.7
143.2
38.1
40.0
7.22 (1H, d, J = 11.0)
6.52 (1H, dd, J = 11.0, 15.0)
6.22 (1H, dt, J = 15.0, 7.5)
2.47, 2.13 (each 1H, m)
1.75 (1H, m)
Sugar analysis
8″
9″
10″
11″
12″
72.1
40.7
173.9
12.8
4.09 (1H, m)
2.54 (2H, m)
Sugar configurations were determined using a previously
described method [16]. Compounds 1 and 2 (each
1.0 mg) were hydrolyzed by heating in 1 mol/L HCl
(0.2 mL) and neutralized with Amberlite IRA400. After
1.94 (3H, s)
0.99 (3H, d, J = 7.0 Hz)
14.3

4
R. HARAMIISHI ET AL.
evaporation, the residues were dissolved in pyridine
(0.2 mL) containing L-cysteine methyl ester hydrochlor-
ide (1.0 mg) and heated at 60°C for 1 h. o-Tolyl isothio-
cyanate (1.0 mg) in pyridine (0.2 mL) was then added to
each mixture and heated at 60°C for 1 h. The reaction
mixtures were directly analyzed by RP-HPLC under
Condition 2. The peaks of derivatives from compound
1 coincided with those of the derivatives similarly pre-
pared from authentic D-glucose. The sugar residue of
compound 2 was confirmed to be D-xylose by similar
way using authentic D-xylose.
performed on day 7 to the LPS, GA-L, and GA-H groups,
and the CON group received saline as the vehicle.
One day after either saline or LPS injection, the mice
were subjected to an open field test to monitor the spon-
taneous behavior of mice, and on the following day, the
mice were sacrificed.
Open field test
The open field device had the following dimensions:
L × W × H of 70 cm × 70 cm × 50 cm. Each mouse
was placed at the center and allowed to move freely
during a 10-min session. The total time of immobility
was analyzed using the ANY-maze Video Tracking
System (Stoelting, Wood Dale, IL, USA) connected to
a USB digital camera.
Partial acid hydrolysis of compound 1
A solution of compound 1 (0.2 mg) was prepared in
H2O (0.2 mL) and 1 mol/L HCl (0.1 mL) and heated
in a boiling water bath for 3 h. The reaction mixture
was analyzed by HPLC (Condition 3) to detect glans-
reginic acid (4) and 4a.
Immunohistochemistry
The mice were transcardially perfused with ice-cold
phosphate-buffered saline when sacrificed, and the
brain was removed and postfixed with 4% paraformalde-
hyde. The brain samples were sectioned to 30-μm-thick
sagittal sections and incubated in 5% normal goat serum
blockade, followed by immunostaining using a rabbit
polyclonal antibody against ionized calcium binding
adaptor molecule 1 (Iba1, 1:1000; Wako, Osaka, Japan)
to stain the microglia. As the secondary antibody,
EnVision-plus system-HRP-labeled polymer (anti-
rabbit; Dako, Glostrup, Denmark) was used. DAB sub-
strate (SK-4100; Vector Laboratories, Burlingame, CA,
USA) was used to develop and visualize immunoreactiv-
ity signals, and images were captured using a microscope
(CX21; Olympus, Tokyo, Japan). The immunoreactive
signals were quantified using ImageJ software.
Isomerization of compounds 3–28
A solution of compound 3 (0.1 mg) in 0.05 mol/L
phosphate buffer (pH 7.4) (0.1 mL) was left standing
at room temperature for 3 h. HPLC (Condition 1) of
the reaction mixture revealed that an isomerized pro-
duct was identical with rugosin C methyl ester (28).
Preparation of test solution of nuts for HPLC
Walnut and other nut samples were ground, dis-
solved in 50% aqueous MeOH (100 mg in 1.0 mL),
sonicated for 5 min, and centrifuged to obtain the
supernatant, which was used as the test solution.
HPLC was performed under Condition 4.
Glansreginin A treatment and systemic injection
of LPS in mice
Statistical analysis
Data of individual groups are expressed as mean
SEM. Data were analyzed using an unpaired t-test or
Dunnett’s multiple comparison test (Prism6;
GraphPad Software, La Jolla, CA, USA). The results
with a p value of < 0.05 were considered significant.
Six-week-old male ICR mice were purchased from
Japan SLC (Hamamatsu, Japan). The mice were
maintained in an animal room under the following
condition: 23°C 1°C and 12-h light/dark cycle. Tap
water and stock diet were freely available to the mice
during the experimental period. All animal experi-
ments were performed following the approved proto-
col 16013 in accordance with the Guidelines for
Animal Experimentation, certified by the Animal
Care and Use Committee of Matsuyama University.
The mice were divided into the following four groups:
control (CON; n = 6), LPS (n = 7), GA-L (n = 6), and GA-
H (n = 6). Oral administration of the vehicle (5%
dimethyl sulfoxide/H2O) was performed to both the
CON and LPS groups for 8 days. Glansreginin A was
dissolved in the vehicle and two doses, 50 mg/kg (GA-L)
and 100 mg/kg (GA-H), were set for 8 days of adminis-
tration. Intraperitoneal LPS treatment (1 mg/kg) was
Results and discussion
Isolation and characterization of constituents
from walnut
An 80% aqueous MeOH extract of walnut was fractio-
nated by chromatography on Diaion HP-20 with
aqueous MeOH, and then each fraction showing
similar patterns on HPLC was combined. Further purifi-
cation was performed by repeated chromatography over
Toyopearl HW-40F, YMC GEL ODS-AQ, Chromatorex
ODS, and Sephadex LH-20 with a step-wise gradient
elution using aqueous MeOH to obtain 30 compounds,

CHARACTERISTIC COMPONENTS IN WALNUT
5
including three compounds (1, 2, and 3) that are unpub-
lished. Compounds 4–30 were identified as glansreginic
acid (4) [5,17], glansreginin A (5) [5], methyl gallate (6),
ellagic acid (7) [18], (+)-catechin (8) [19], blumenol
C glucoside (9), byzantionoside B (10) [20], 4′-dihydro-
phaseic acid β-glucopyranose ester (11) [21], 5-hydroxy-
tryptamine (12) [22], valoneic acid dilactone methyl ester
(13) [23], ellagic acid 4-O-β-D-xyloside (14) [24], glans-
reginic acid 8-O-β-D-glucoside (15) [25], glansreginin B
(16) [5], 1,2,6-tri-O-galloyl-β-D-glucose (17), 1,2,4,6-
tetra-O-galloyl-β-D-glucose (18), 1,2,3,4,6-penta-O-gal-
loyl-β-D-glucose (19) [26], strictinin (20), isostrictinin
(21) [27], tellimagrandin I (22), tellimagrandin II (23)
[28], casuarictin (24), pedunculagin (25) [29], pterocar-
inin C (26) [30], rugosin C (27) [31], rugosin C methyl
ester (28), casuarinin (29) [29], and euprostin A (30) [32]
(Figure 1). To the best of our knowledge, compounds 4,
9–11, 15, and 26 were first obtained from walnut in this
study.
Compound 1 was isolated as a brown amorphous
powder. Its molecular formula C44H52O21N2 was deter-
mined based on HR-ESI-MS data (m/z 943.2973 [M-H]−)
and the 13C-NMR spectrum displaying 44 13C signals.
The UV spectrum showed the absorption maxima at
Figure 1. Structure of compounds 1–30. G, galloyl group; (S)-HHDP, (S)-hexahydroxydiphenoyl group.

6
R. HARAMIISHI ET AL.
213 and 264 nm. The 1H-NMR spectrum of 1 (Table 1)
displayed signals attributed to two sets of 1,2-disubsti-
tuted benzene, isolated methylene protons [δ 6.86,
6.91, 7.03, 7.05, 7.28, 7.31, 7.39 (2H), and 3.17 (4H)],
and a β-glucopyranose moiety, which was supported by
information in the 1H-1H correlation spectroscopy
(COSY) and heteronuclear single quantum coherence
(HSQC) spectra. In addition, the signals due to mutually
coupled olefin protons (δ 6.22, 6.52, and 7.22), methylene
[δ 2.13, 2.47, 2.54 (2H)], methine (δ 1.75 and 4.09), and
methyl (δ 0.99) protons, along with a vinyl methyl proton
(δ 1.94) were observed. These spectral features were
similar to those of glansreginin A (5) [5], except for the
presence of an extra 2-quinolone glucoside moiety (unit
A in Figure 2). Therefore, compound 1 was predicted to
have a structure as shown in formula 1. The 13C-NMR
spectrum of 1 (Table 1) was consistent with the presumed
structure. The structure (1) was chemically substantiated
by its acid hydrolysis yielding glansreginic acid (4) and 4a
as reported those of glansreginin A [5]. D-Series of glu-
cose as sugar units of 1 was determined by acid hydrolysis
followed by HPLC analysis of derivatives prepared by
a reaction with L-cysteine methyl ester and o-tolyl iso-
thiocyanate according to the previously reported method
[16]. The presence of the assumed units was further
supported by correlations in HSQC and heteronuclear
multiple bond connectivity (HMBC) spectra. The linking
position for each unit was determined using HMBC
analysis, which showed three-bond correlations among
glucose H-1, 1′ (δ 4.14, 4.23)/C-4, 4′ (δ 79.4, 79.5), and
glucose H-6, 6′ (δ 4.09, 4.19)/C-1″, 10″ (δ 170.1, 173.9) as
shown in Figure 2. Two β-glycosidic linkages in the
glucose cores were evidenced by large coupling constants
(each J= 8.0 Hz) of anomeric proton signals. The absolute
configuration at C-4, 4′, and C-7″, as well as those of
compound 1 remains undetermined [5]. On the basis of
these data, the structure of compound 1 was established
as shown in formula 1, and named glansreginin C.
Compound 2 was found to have the molecular for-
mula C26H18O16 by HR-ESI-MS, m/z 580.0522 [M-H]‾.
The UV spectrum showed the absorption maxima at
343, 275, and 213 nm, which were characteristic of
ellagic acid derivatives. The 1H- and 13C-NMR spectra
of 2 also showed the signals assignable to ellagic acid
moiety [2 aromatic 1H-singlets (δ 7.74, 7.47), 12 aro-
matic carbons, and 2 carbonyl carbons (δ 107.3, 109.9,
111.7, 113.6, 113.7, 116.8, 138.0 (2C), 141.4, 145.4,
149.0, 150.1, 161.4, and 161.6)]. Furthermore, the pre-
sence of a galloyl group and pentose residue was indi-
cated by one aromatic 2H-singlet (δ 7.16), six aromatic
carbons, and one carbonyl carbon [δ 110.5 (2C), 121.7,
139.8, 146.4 (2C), and 168.2] and five sugar carbons in
the range of δ 67.1–104.3 and sequentially coupled five
proton signals at δ 5.21–3.58. The presence of ellagic
acid and galloyl residues was further supported by cor-
relations in the HSQC and HMBC spectra. Pentose
1
proton signals assigned by the H-1H COSY spectrum
and 13C-NMR signals were similar to those of xylose.
D-Xylose as sugar units of 2 was confirmed by acid
hydrolysis followed by HPLC analysis of derivatives
prepared in the reported way [16]. The β-glycosidic
linkage in the xylose core was evidenced by large cou-
pling constants (J = 7.5 Hz) of the anomeric proton. The
linking position for each unit was determined using
HMBC, which showed three-bond correlations among
the xylose anomeric proton H-1 (δ 5.07)/ellagic acid
unit A
2
unit A
Key HMBC correlation
Key ROESY correlation
1
Figure 2. Key heteronuclear multiple bond connectivity (HMBC) correlations and rotating frame nuclear Overhauser effect
spectroscopy (ROESY) correlation of compounds 1 and 2.

CHARACTERISTIC COMPONENTS IN WALNUT
7
unit C-4 (δ 149.0) and the xylose H-3 (δ 5.21)/galloyl
group C-7‴ (δ 168.2). Furthermore, the rotating frame
nuclear Overhauser effect spectroscopy (ROESY) spec-
trum showed the anomeric proton H-1 (δ 5.07) corre-
lated with H-5 (δ7.74) suggesting the spatial proximity
of the two protons (Figure 2). Therefore, compound 2
was established as ellagic acid 4-O-(3′-O-galloyl)-β-
D-xyloside.
Compound 3 was suggested to be an ellagitannin
as revealed by UV, 1H- and 13C-NMR spectra.
Aromatic proton signals characteristic of galloyl
group (δ 7.16, 2H, s) and hexahydroxydiphenoyl
(HHDP) and tergalloyl groups [δ 6.87, 6.59, 6.57,
6.46 and 6.39 (each 1H, s)] together with signal
pattern of glucopyranose residue were reminiscent
of similarity to those of platycaryanin A (3a) [33],
which has been reported to co-occur in walnuts [5].
A distinguishable feature between 3 and 3a in the
spectra was the presence of a methoxy signal (δH 3.82
and δC 52.9) in the former suggested that compound
3 is platycaryanin A methyl ester (3). The presumed
structure 3 was confirmed by isomerization (Smiles
rearrangement) of the tergalloyl group into valoneoly
group under mild condition [34] affording rugosin
C methyl ester (28). Although this compound is
unpublished, it may be an artifact produced during
the extraction or isolation procedure with using
MeOH [35]. To confirm that this compound was an
artifact, walnut material was extracted using 70%
aqueous acetone, and then HPLC analysis was per-
formed. As a result, the peak of compound 3 was
detected in the aqueous acetone extract, therefore it
was regarded as a natural product.
Component comparison of nut extracts by HPLC
To clarify the characteristic components of walnuts,
RP-HPLC of walnut extract was compared with those
of six nut (almond, pistachio, hazelnut, cashew nut,
pecan nut, and macadamia nut) extracts. The results
of HPLC analysis revealed that a peak with a retention
time of 25 min, a corresponding peak to glansreginin
A (5), was the main peak in walnut extract, but this
peak was not observed in any other extracts (Figure 3).
Figure 3. HPLC profiles of nut extracts prepared with 50% aqueous MeOH.
(a) HPLC (detected at 280 nm) chromatograms of nut extracts (a: walnut, b: almond, c: pistachio, d: hazelnut, e: cashew nut, f: pecan nut, and g:
macadamia nut). (b) Three-dimensional chromatogram (detected at 200–400 nm) of the walnut extract.

8
R. HARAMIISHI ET AL.
Previous studies have identified glansreginin A not
only in walnuts (Juglans regia) [5] but also in hazelnuts
and pecan nuts using LC/MS/MS [36,37]. As shown in
Figure 3, the peak equivalent to glansreginin A was not
recognized in the extracts of hazelnuts and pecan nuts
by HPLC in this study, suggesting that glansreginin
A might be a minor constituent which might be scar-
cely detected by LC/MS/MS. On the contrary, the peak
equivalent to glansreginin A was commonly observed
by HPLC in every commercial walnut we checked (data
not shown), indicating that glansreginin A might be an
indicator component in walnuts.
Total time immobile
100
80
60
40
20
0
#
##
**
CON
LPS
GA-L
GA-H
Anti-inflammatory effect of glansreginin A in vivo
Figure 4. Total time of immobility in the open field test.
The opened, closed, shaded, and hatched bars represent CON, LPS,
Walnuts are known to prevent and improve risk
factors associated with lifestyle diseases, and fatty
acids and polyphenols among its components have
been suggested to contribute to its activities [4–13].
On the contrary, recent studies showed that the
dietary supplementation of walnuts could improve
memory deficits and learning skills in transgenic
mouse model of AD, and the contribution of fatty
acids and polyphenols is also discussed [14]. As
fatty acids and polyphenols are widely distributed
in plant materials, we evaluated whether glansregi-
nin A (5), a supposed indicator component in wal-
nuts, has the ability to exert the neuroprotective
effect in the brain.
In this study, we perorally administered of glans-
reginin A to LPS-induced systemic inflammatory
model mice. Inflammatory response is an extremely
important mechanism in the human body to pro-
tect from infection/injury, but hyper-inflammation
triggers chronic diseases in not only peripheral
tissues but also brain, including neurodegenerative
diseases such as AD [38,39]. LPS, an endotoxin of
gram-negative bacteria, is often used in research to
elicit inflammation, and systemic injection of LPS
is known to cause hyperactive microglial response
and IL-1β level elevation in the brain, resulting in
cognitive dysfunction [40] and sickness behavior in
mice [41].
GA-L, and GA-H, respectively. The values are mean
SEM. The
symbols indicate significant difference for the following conditions:
CON vs. LPS (**p < 0.01), LPS vs. GA-L (##p < 0.01), and LPS vs. GA-H
(#p < 0.05).
immune cells in brain. Figure 5(a) shows that 1) the
morphology of microglia in the hippocampus of the
LPS group was changed to the activated form (ame-
boid form); 2) the LPS-induced activation of micro-
glia was significantly suppressed in both the GA-L
and GA-H groups, compared with the morphology of
CON group (ramified form). Figure 5(b) shows that
intensity of the immune-positive signals in the LPS
group was significantly (##p < 0.01) decreased by GA-
H-administration and slightly (p = 0.056) decreased
by GA-L-administration. These results suggested that
glansreginin A in walnuts might have a strong anti-
inflammatory effect. To the best of our knowledge,
this is the first study to show that glansreginin A has
neuroprotective effects.
Inflammation is a physiological response to infec-
tion/injury; however, excessive inflammatory reac-
tions are the key contributor to the pathophysio-
logical process of chronic diseases including various
intracerebral diseases such as age-related neurologi-
cal disorders, AD, multiple sclerosis, and brain
tumors [42]. Among various cell types in the brain
(neurons, astrocytes, microglia, oligodendrocytes,
pericytes, amongst others), microglia contribute the
most to neuronal dysfunction and death during
neuroinflammation, because they overproduce
inflammatory mediators such as interleukin-1β,
tumor necrosis factor-α, and nitric oxide [43]. The
present findings taken together with the studies
described above suggest that glansreginin A can
exert a neuroprotective effect via anti-inflammation
in the brain. Walnuts containing glansreginin
A might be recognized as functional food for brain
diseases in the near future.
To investigate the effect of glansreginin A
administration on locomotive behavior, the mice
were subjected to an open field test. As shown in
Figure 4, LPS-injected mice exhibited significantly
**
(
p < 0.01) higher locomotive activity than that of
the CON group, and this phenomenon was sup-
pressed by glansreginin A administration; the GA-
L group (##p < 0.01) and GA-H group (#p < 0.05).
Next, the mice were sacrificed at the end of the
experiment period, and the brain sections were sub-
jected to immunohistochemical analysis with the Iba1
antibody, a specific marker of microglia, resident

CHARACTERISTIC COMPONENTS IN WALNUT
9
CON
GA-L
LPS
GA-H
##
p=0.056
**
6000
4000
2000
0
CON
LPS
GA-L
GA-H
Figure 5. Effects of glansreginin A (5) on Iba1 immunoreactivity in the hippocampus.
(a) Sagittal sections were stained with the anti-Iba1 antibody. Scale bar = 100 μm. (b) Quantitative analysis of Iba1-positive signals using ImageJ
software. The opened, closed, shaded, and hatched bars represent CON, LPS, GA-L, and GA-H, respectively. The values are mean SEM. The symbols
indicate significant difference for the following conditions: CON vs. LPS (**p < 0.01) and LPS vs. GA-H (##p < 0.01).
It is well known that walnut is a highly nutri-
tious food. This study provides valuable informa-
tion about the benefits of walnut on brain health
and function. This study also presents a simple
method to evaluate the quality of walnut products
using an indicator.
Author contributions
R.H., S.O. and Y.A. conceived and designed the experiments.
R.H., S.O., M.Y., M.N., Y.F. and Y.A. performed the experi-
ments or helped the experimental techniques. H.I. supervised
the experimental techniques. R.H., S.O. and Y.A. wrote the
paper. Y.F. and Y.A. reviewed manuscript. All authors have
read and approved the submitted manuscript.
Acknowledgments
Disclosure statement
A portion of this work was supported by JSPS KAKENHI Grant
Number JP26350168. This work was supported by the Nagai
Memorial Research Scholarship from the Pharmaceutical
Society of Japan.
No potential conflict of interest was reported by the authors.

10
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