Coixlachryside B: a new benzoxazinoid glycoside from the roots of Coix lachryma-jobi var. ma-yuen (Gramineae)

Article abstract of DOI:10.1080/10286020.2018.1497016

Coix lachryma-jobi L. var. ma-yuen has been a source of food and traditional folk medicine in some parts of Asia for thousands of years; however, the roots of this plant have not been phytochemically investigated. Herein, we report the isolation of a new benzoxazinoid glycoside, coixlachryside B (1), along with ten known compounds (2–11), from the roots of C. lachryma-jobi var. ma-yuen using a variety of chromatographic methods. Among the known compounds, the absolute configuration of compound 4 was determined. The structures of all compounds were elucidated by interpreting NMR spectroscopic data, and experimental and calculated electronic circular dichroism spectra.

Full text of DOI:10.1080/10286020.2018.1497016

Journal of Asian Natural Products Research
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Coixlachryside B: a new benzoxazinoid glycoside
from the roots of Coix lachryma-jobi var. ma-yuen
(Gramineae)
Yun-Hyeok Choi, Chun Whan Choi, Seong Hyun Hong, Se Kyu Park, Joa Sub
Oh, Dongho Lee & Seong Su Hong
To cite this article: Yun-Hyeok Choi, Chun Whan Choi, Seong Hyun Hong, Se Kyu Park, Joa
Sub Oh, Dongho Lee & Seong Su Hong (2018): Coixlachryside B: a new benzoxazinoid glycoside
from the roots of Coixꢀlachryma-jobi var. ma-yuen (Gramineae), Journal of Asian Natural Products
Research, DOI: 10.1080/10286020.2018.1497016
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JOURNAL OF ASIAN NATURAL PRODUCTS RESEARCH
https://doi.org/10.1080/10286020.2018.1497016
Coixlachryside B: a new benzoxazinoid glycoside from the
roots of Coix lachryma-jobi var. ma-yuen (Gramineae)
Yun-Hyeok Choia,b, Chun Whan Choia, Seong Hyun Honga, Se Kyu Parkc,
a
Joa Sub Ohd, Dongho Leeb and Seong Su Hong
ꢀ
ꢀ
aBio-Center, Gyeonggido Business & Science Accelerator (GBSA), Suwon 16229, Republic of Korea;
bDepartment of Biosystems and Biotechnology, College of Life Sciences and Biotechnology, Korea
c
University, Seoul 02841, Republic of Korea; Hansolbio Co., Ltd, Seongnam 13215, Republic of Korea;
dCollege of Pharmacy, Dankook University, Cheonan 31116, Republic of Korea
ABSTRACT
ARTICLE HISTORY
Received 31 July 2017
Revised 2 July 2018
Accepted 3 July 2018
Coix lachryma-jobi L. var. ma-yuen has been a source of food and
traditional folk medicine in some parts of Asia for thousands of
years; however, the roots of this plant have not been phytochemi-
cally investigated. Herein, we report the isolation of a new ben-
zoxazinoid glycoside, coixlachryside B (1), along with ten known
compounds (2–11), from the roots of C. lachryma-jobi var. ma-
yuen using a variety of chromatographic methods. Among the
known compounds, the absolute configuration of compound 4
was determined. The structures of all compounds were elucidated
by interpreting NMR spectroscopic data, and experimental and
calculated electronic circular dichroism spectra.
KEYWORDS
Gramineae; Coix lachryma-
jobi var. ma-yuen;
benzoxazinoid; coixlachry-
side; phenolic compounds
1. Introduction
Coix lachryma-jobi L. var. ma-yuen Stapf. (Gramineae), commonly known as adlay or
Job’s tears, is a miscellaneous cereal that has been an important food item in some
parts of East and Southeast Asia for thousands of years [1]. Coix seeds have been
used as a traditional folk medicine for relieving heat, inducing diuresis, assisting pus
drainage, stimulating lung and spleen function, and treating arthritis and diarrhea
[1,2]. C. lachryma-jobi var. ma-yuen has been reported to have several chemical con-
stituents, including benzoxazinoids, lactams, lignans, phenolic acids, flavonoids, fatty
acids, and phytosterols [3–9]. The chemical constituents of Coix contribute to its
range of medical and nutritional benefits. Some of these compounds have shown a
wide range of biological activities, including immunomodulatory, anti-cancer, antioxi-
dant, anti-inflammatory, anti-proliferative, and gastroprotection activities [7–12]. The
aim of this study was to phytochemically investigate the roots of C. lachryma-jobi
var. ma-yuen. Herein we report the isolation of a new benzoxazinoid glycoside, (2R)-
2-O-(6-acetyl-b-D-glucopyranosyl)-7-methoxy-1,4(2H)-benzoxazin-3-one (1), as well
CONTACT Dongho Lee
dongholee@korea.ac.kr; Seong Su Hong
bestgene@hotmail.com
Supplemental data for this article can be accessed at 10.1080/10286020.2018.1497016.
ß 2018 Informa UK Limited, trading as Taylor & Francis Group

2
Y.-H. CHOI ET AL.
Figure 1. Structures of compounds 1–4 isolated from C. lachryma-jobi var. ma-yuen.
as 10 known compounds (2–11, Figure 1). Among the 10 known compounds, the
absolute stereochemistry of one previously isolated phenolic derivative was identified
for the first time in this study.
2. Results and discussion
Compound 1 was obtained as a colorless amorphous powder, and its molecular for-
mula was determined to be C17H21NO10 by HRESIMS (m/z 400.1254 [M þ H]þ),
1
which requires the molecule to have eight degrees of unsaturation. The H and 13C
NMR data for 1 (Table 1) suggested the presence of one ABX-type aromatic spin sys-
tem [dH 6.83 (1H, d, J ¼ 9.1 Hz, H-5), 6.60 (1H, d, J ¼ 2.1 Hz, H-8), and 6.59 (1H, dd,
J ¼ 9.1, 2.1 Hz, H-6); dC 156.0, 141.6, 120.3, 116.3, 108.8, and 104.4], an aromatic
OCH3 group [dH 3.68 (3H, s, 7-OCH3); dC 55.8], an acetoxyl group [dH 2.09 (3H, s,
COOCH3); dC 170.8 and 21.2], an acetal methine moiety [dH 5.60 (1H, s, H-2); dC
1
96.2], and an amine [dH 10.8 (1H, s, NH)]. In addition, the H and 13C NMR spectra
revealed sets of signals corresponding to a b-glucopyranosyl group [dH 4.62 (1H, d,
J ¼ 8.4 Hz, H-10); dC 103.7 (C-10), 73.5 (C-20), 76.8 (C-30), 70.1 (C-40), 74.5 (C-50),
64.2 (C-60)] (Table 1). The 13C NMR spectrum of 1 was almost superimposable onto
that of HMBOA-Glc (2) except for the presence of one acetoxyl group. The correl-
ation between H-60 and COOCH3 observed in the HMBC spectrum of 1 establishes
that the acetoxyl moiety was located at C-60. The free sugar was confirmed to be D-
glucose after acid hydrolysis, optical rotation measurement, and TLC comparison
with an authentic sample. In addition, the b-configuration of the sugar moiety was
determined from the 1H-NMR coupling constant of its anomeric proton
(J1,2 > 7.0 Hz) [13], while the C-2 connectivity of the sugar was determined on the
basis of HMBC correlations between Glc H-10 (dH 4.62) and C-2 (dC 96.2) (Figure 2).
The absolute configuration of C-2 was confirmed as being 2R by the positive optical-
25
rotation ([a]D þ44.5) and the positive Cotton effects (De þ14.8) observed at
216 nm in the circular dichroism (CD) spectrum of 1 [3]. Therefore, the structure of
compound 1 was determined to be (2R)-2-O-(6-acetyl-b-D-glucopyranosyl)-7-
methoxy-1,4(2H)-benzoxazin-3-one, with the trivial name “coixlachryside B.”
Compound 4 was obtained as a white amorphous powder that exhibited a quasi-
molecular ion peak at m/z 379.13971 [M þ H]þ by positive-ion HRESIMS, which cor-
responds to the molecular formula C19H22O8. The 1- and 2-D NMR data for 4 are in
agreement with those of the previously reported tarennone (1,2-bis(3,5-dimethoxy-4-
hydroxyphenyl)-3-hydroxy-propan-1-one) isolated from the whole plant of Tarenna

JOURNAL OF ASIAN NATURAL PRODUCTS RESEARCH
3
Table 1. 1H- and 13C-NMR spectroscopic data of compounds 1 and 4.a
1b
4c
Position
dH mult. (J Hz)
dC mult.
Position
dH mult. (J Hz)
7.35 s
dC mult.
2
3
5
6
7
8
9
10
10
20
30
40
50
60
5.60 s
96.2 d
160.0 s
116.3 d
108.8 d
156.0 s
104.4 d
141.6 s
120.3 s
103.7 d
73.5 d
76.8 d
70.1 d
74.5 d
64.2 t
1
2
3
4
5
6
7
8
9
129.0 s
107.8 d
148.9 s
142.3 s
148.9 s
107.8 d
199.4 s
6.83 d (9.1)
6.59 dd (9.1, 2.1)
6.60 d (2.1)
7.35 s
4.75 dd (8.4, 4.9)
4.26 dd (10.5, 8.4)
3.73 dd (10.5, 4.9)
56.8d
d
65.4 t
4.62 d (8.4)
2.91 t (9.1)
3.16 t (9.1)
3.02 t (9.1)
3.42 m
4.31 dd (11.9, 2.1)
4.01 dd (11.9, 2.1)
10.8 s
10
20
30
40
50
60
129.3 s
6.63 s
106.6 d
149.6 s
136.0 s
149.6 s
106.6 d
NH
6.63 s
3.85 s
3.80 s
7-OCH3
COOCH3
COOCH3
3.68 s
55.8 s
170.8 s
21.2 q
3,5-OCH3
56.8d
56.7 s
s
30,50-OCH3
2.09 s
aPeaks were assigned by analyses of the 1D and 2D NMR spectra (Data recorded at 700 and 175 MHz).
bMeasured in DMSO-d6.
cMeasured in CD3OD.
dOverlapping signals.
Figure 2. Selected COSY and HMBC correlations of compounds 1 and 4.
attenuata [14], but whose absolute stereochemistry was not assigned. Hence, the
absolute configuration of 4 was determined by comparing experimental and calcu-
lated electronic circular dichroism (ECD) data, which is a method that has been
applied for determining the absolute configurations of natural metabolites [15].
Following systematic conformational searching, geometry optimizations, and ECD
calculations, the ECD spectrum was generated using the SpecDis 1.64 software. The
calculated ECD spectrum of 4 (Figure 3) closely matches the experimental spectrum,
which suggests the 8S absolute configuration; consequently, 4 was determined to be
(8S)-tarennone, as shown.
The remaining compounds were determined to be (2R)-2-hydroxy-7-methoxy-
1,4(2H)-benzoxazin-3-one glucoside (HMBOA-Glc, 2) [3], (2R)-2-hydroxy-4,7-dime-
thoxy-1,4(2H)-benzoxazin-3-one glucoside (HDMBOA-Glc, 3) [16], 8R-evofolin B (5)
[17], 3,4,5-trimethoxybenzyl alcohol (6) [18], a-hydroxypropiosyringone (7) [19],
b-hydroxypropiovanillone (8) [20], b-hydroxypropiosyringone (9) [21], 1-O-feruloyl-
glycerol (10) [22], and 10Z,13Z-nonadecadienoic acid (11) [23]; their chemical

4
Y.-H. CHOI ET AL.
Figure 3. Calculated and experimental ECD spectra of compounds 4 and 5.
structures were elucidated by comparing their spectroscopic and MS data with those
reported in the literature. To the best of our knowledge, compounds 4–11 were iso-
lated for the first time from the Coix genus.
3. Experimental
3.1. General experimental procedures
Optical rotations were measured using a JASCO P-2000 polarimeter, and CD spectra
were recorded on a JASCO J-715 spectropolarimeter (Tokyo, Japan). NMR spectra
were acquired on a Bruker Ascend III 700 spectrometer (Rheinstetten, Germany) using
DMSO-d6 or CD3OD as solvent. HRESIMS was performed on Triple TOF 5600þ
mass spectrometer (AB SCIX, Foster City, CA, USA). Open column chromatography
was performed using ODS-A (12 nm S-7 mm, YMC GEL, Kyoto, Japan). Thin-layer
chromatography was performed using precoated silica gel 60 F254 (0.25 mm, Merck)
plates, and spots were detected using a 10% aqueous vanillin-H2SO4 spray reagent.
Preparative HPLC was performed on a Shimadzu (LC-8A pump and SPD-20A UV/
VIS detector) using a YMC-Pack ODS A column (250 ꢁ 20 mm I.D.), with a mixed
MeCN/H2O solvent system at a flow rate of 10 ml/min. MPLC (Combi Flash RF,
R
V
Teledyne ISCO, Lincoln, NE, USA) separations were performed using a RediSep Rf
silica-gel column. All other chemicals and reagents were of analytical grade.
3.2. Plant material
The roots of Coix lachryma-jobi var. ma-yuen were collected in Yeoncheon-gun,
Gyeonggi-do, Korea, in November 2013 and identified by one of the authors (Prof. J.
S. Oh). A voucher specimen (G54) was deposited in the herbarium of the Bio-Center,
Gyeonggido Business & Science Accelerator, Suwon, Korea.

JOURNAL OF ASIAN NATURAL PRODUCTS RESEARCH
5
3.3. Extraction and isolation
The dried roots of C. lachryma-jobi var. ma-yuen (1.9 kg) were extracted with 70%
ethanol (2 ꢁ 14 L) at room temperature. The combined ethanol extracts were then
concentrated in vacuo at 40 ꢂC to yield 333.3 g of a residue. The ethanol extract was
suspended in distilled water and then partitioned successively with n-hexane, CH2Cl2,
EtOAc, and n-BuOH, respectively. The EtOAc-soluble layer (5.5 g) was separated
using a Diaion HP-20 column chromatography (CC) eluted with a H2O/MeOH
(20:80–0:100) step gradient to give seven fractions (G54 ꢃ 6ꢃ1 to G54 ꢃ 6ꢃ7).
Fraction G54 ꢃ 6ꢃ3 (1.2 g) was further passed through an RP-18 MPLC column
R
(RediSepV Rf silica gold 430 g, 150 ml/min) eluted with a H2O/MeCN (95:5–50:50,
50 min) step gradient to give eight fractions (G54 ꢃ 7ꢃ1 to G54 ꢃ 7ꢃ8). G54 ꢃ 7ꢃ4
(120 mg) was purified by preparative HPLC (YMC-Pack ODS-A, 5 lm, 250 ꢁ 10 mm
I.D., MeCN/H2O, 10:90–20:80, flow rate 12.0 ml/min) to yield compounds 8
(tR ¼ 14 min, 18.6 mg) and 9 (tR ¼ 12 min, 2.1 mg). G54 ꢃ 7ꢃ6 (164 mg) was purified
further by preparative HPLC (MeCN/H2O, 15:85 isocratic mode, flow rate 12.0 ml/
min) to afford compounds 2 (tR ¼ 14 min, 39.6 mg), 3 (tR ¼ 16 min, 6.4 mg), and 7
(tR ¼ 20 min, 1.0 mg). Subfraction G54 ꢃ 7ꢃ2 (216 mg) was subjected to reversed-
phase C18 CC and eluted with MeOH/H2O (10:90 to 50:50) to obtain eight fractions
(G54 ꢃ 9ꢃ1 to G54 ꢃ 9ꢃ8). Fraction G54 ꢃ 9ꢃ2 (65 mg) was purified by preparative
HPLC (MeCN/H2O, 15:85 isocratic mode, flow rate 12.0 ml/min) to afford com-
pounds 1 (tR ¼ 17 min, 7.8 mg) and 6 (tR ¼ 14 min, 4.0 mg). Fraction G54 ꢃ 9ꢃ5
(74 mg) was purified further by preparative HPLC (MeCN/H2O, 25:75 isocratic mode,
flow rate 12.0 ml/min) to afford compounds 4 (tR ¼ 10 min, 3.2 mg), 5 (tR ¼ 17 min,
3.6 mg), and 10 (tR ¼ 13 min, 4.5 mg). Fraction G54 ꢃ 6ꢃ5 (1.3 g) was separated by
R
MPLC with a silica-gel column (RediSepV Rf silica gold 80 g, 85 ml/min) and eluted
with a CHCl3/MeOH gradient system (1:0–0:1) to give seven fractions (G54 ꢃ 10 ꢃ 1
to G54 ꢃ 10 ꢃ 7). G54 ꢃ 10 ꢃ 2 (380 mg) was further purified by preparative HPLC
(MeOH/H2O, 95:5–100:0, flow rate 8.0 ml/min) to yield compound 11
(tR ¼ 21 min, 8 mg).
3.3.1. (2R)-2-O-(6-Acetyl-b-D-glucopyranosyl)-7-methoxy-1,4(2H)-benzoxazin-3-one
(coixlachryside B, 1)
25
Colorless amorphous powder; [a]D þ44.5 (c 0.8, MeOH); CD (MeOH) De216 nm
1
þ14.8; H-NMR (700 MHz, DMSO-d6) and 13C-NMR (175 MHz, DMSO-d6) spectral
data see Table 1; ESIMS: m/z 422 [M þ Na]þ, 369 [M – H]–; HRESIMS (positive
mode): m/z 400.1245 [M þ H]þ (calcd for C17H22NO10, 400.1238).
3.3.2. 8S-Tarennone (4)
25
White amorphous powder; [a]D þ21.3 (c 0.5, MeOH); CD (MeOH) De237 nm þ4.64,
De268 nm ꢃ1.97, De311 nm þ2.75; 1H-NMR (700 MHz, CD3OD) and 13C-NMR
(175 MHz, CD3OD) spectral data see Table 1; ESI-MS: m/z 379 [M þ H]þ, 401
[M þ Na]þ, 779 [2M þ Na]þ, 377 [M – H]ꢃ; HRESI-MS (positive ion mode): m/z
379.1397 [M þ H]þ (calcd for C19H23O8, 379.1387).

6
Y.-H. CHOI ET AL.
3.4. Acid hydrolysis of 1, and absolute configuration determination of sugar
A solution of compound 1 (3 mg) in 2M HCl (MeOH/H2O 4:1, 5 ml) was refluxed at
90 ꢂC for 6 h. After cooling, the reaction mixture was diluted with H2O (20 ml) and
neutralized with NaHCO3, followed by extraction with CHCl3 (3 ꢁ 20 ml). The aque-
ous layer was concentrated to an appropriate volume (1 ml) and examined by TLC
(silica gel) (CHCl3/MeOH/H2O solvent system, 65:35:10) for sugar analysis. The Rf
value of the D-glucose spot was 0.25, which was detected by spraying with vanillin-
sulfuric acid followed by heating. The specific rotation of the free sugar obtained
25
from the aqueous residue was as follow: [a]D þ47 (c 0.05, H2O) of D-glucose from
1 [24].
3.5. Computational methods
Starting with the conformation of compound 4 created by Chem3D modeling, a con-
formational search was first performed using Chem3D with the MMFF94 force field,
and several conformers were selected for further geometry optimization. Geometries
were optimized and reoptimized at the B3LYP/6–31 þ G(d,p) level using the Gaussian
09 software package. TDDFT CD calculations were performed at the CAM–B3LYP/
SVP level on the optimized conformers using a CPCM solvent model in methanol.
The calculated CD spectra were simulated and generated with a half-bandwidth of
0.3 eV using SpecDis 1.64 software [25]. The CD curve of the compound was
Boltzmann weighted follow UV correction.
Disclosure statement
No potential conflict of interest was reported by the authors.
Funding
This work was financially supported by Korea Institute of Planning and Evaluation for
Technology in Food, Agriculture, Forestry and Fisheries (IPET) through Agri-Bio industry
Technology Development Program, funded by Ministry of Agriculture, Food and Rural Affairs
(MAFRA) (114144-3).
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