Structure determination and quantification of a new flavone glycoside with anti-acetylcholinesterase activity from the herbs of Elsholtzia ciliata

Article abstract of DOI:10.1080/14786419.2017.1413556

Three acacetin triglycosides (compounds 1, 2 and 3) were isolated from the herbs of Elsholtzia ciliata (Labiatae). The structure were identified as 7-O-β-D-glucopyranosyl-(1?→?2)[α-L-rhamnopyranosyl-(1?→?6)]-β-D-glucopyranoside (compound 1), 7-O-(6-O-acetyl)-β-D-glucopyranosyl-(1?→?2)[α-L-rhamnopyranosyl-(1?→?6)]-β-D-glucopyranoside (compound 2) and 7-O-(6-O-acetyl)-β-D-glucopyranosyl-(1?→?2)[(4-O-acetyl)-α-L-rhamnopyranosyl-(1?→?6)]-β-D-glucopyranoside (compound 3) of acacetin. The structures of these compounds were determined on the basis of 2D-NMR spectroscopic data. Compound 3 has not been isolated from a natural source. In addition, the three compounds were quantitatively analysed by HPLC. Acetylcholinesterase (AChE) inhibition activity was assayed to find anti-Alzheimer’s activity, since this enzyme increases the concentration of acetylcholine (ACh), a neurotransmitter, responsible for brain’s memory. Acacetin, the aglycone of the three compounds, exhibited a potent anti-cholinesterase activity (IC₅₀, 50.33?±?0.87), though its glycosides (1, 2 and 3) were less active. HPLC analysis demonstrated that the three compounds were contained in the MeOH extract in the order of compounds 2 (12.63?mg/g extract) > 3 (3.10?mg/g) > 1 (2.92?mg/g).

Full text of DOI:10.1080/14786419.2017.1413556

Natural Product Research
Formerly Natural Product Letters
ISSN: 1478-6419 (Print) 1478-6427 (Online) Journal homepage: http://www.tandfonline.com/loi/gnpl20
Structure determination and quantification
of a new flavone glycoside with anti-
acetylcholinesterase activity from the herbs of
Elsholtzia ciliata
Agung Nugroho, Jong-Hyuk Park, Jae Sue Choi, Kyoung-Sik Park, Joon-Pyo
Hong & Hee-Juhn Park
To cite this article: Agung Nugroho, Jong-Hyuk Park, Jae Sue Choi, Kyoung-Sik Park, Joon-
Pyo Hong & Hee-Juhn Park (2017): Structure determination and quantification of a new flavone
glycoside with anti-acetylcholinesterase activity from the herbs of Elsholtzia ciliata, Natural Product
Research, DOI: 10.1080/14786419.2017.1413556
To link to this article: https://doi.org/10.1080/14786419.2017.1413556
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Date: 12 December 2017, At: 08:22

Natural Product research, 2017
ꢁꢁpꢂ://ꢃꢄi.ꢄꢅg/10.1080/14786419.2017.1413556
ꢀ
Structure determination and quantification of a new flavone
glycoside with anti-acetylcholinesterase activity from the
herbs of Elsholtzia ciliata
Agung Nugrohoa , Jong-Hyuk Park , Jae Sue Choi , Kyoung-Sik Parkd,
Joon-Pyo Hong and Hee-Juhn Park
b
c
e
e
ꢆ
b
dꢇpꢆꢅꢁmꢇnꢁ ꢄf agꢅꢄ-Inꢃꢈꢂꢁꢅiꢆꢉ tꢇꢊꢀnꢄꢉꢄgy, lꢆmbꢈng Mꢆngkꢈꢅꢆꢁ univꢇꢅꢂiꢁy, Bꢆnjꢆꢅbꢆꢅꢈ, Inꢃꢄnꢇꢂiꢆ; cꢄꢉꢉꢇgꢇ
ꢊ
ꢄf Mꢇꢃiꢊinꢇ, hꢆꢉꢉym univꢇꢅꢂiꢁy, cꢀꢈnꢊꢀꢇꢄn, rꢇpꢈbꢉiꢊ ꢄf Kꢄꢅꢇꢆ; dꢇpꢆꢅꢁmꢇnꢁ ꢄf Fꢄꢄꢃ Nꢈꢁꢅiꢁiꢄn, Pꢈkyꢄng
Nꢆꢁiꢄnꢆꢉ univꢇꢅꢂiꢁy, Bꢈꢂꢆn, rꢇpꢈbꢉiꢊ ꢄf Kꢄꢅꢇꢆ; dꢇpꢆꢅmꢇnꢁ ꢄf oꢅiꢇnꢁꢆꢉ Mꢇꢃiꢊinꢇ, sꢆngji univꢇꢅꢂiꢁy, Wꢄnjꢈ,
rꢇpꢈbꢉiꢊ ꢄf Kꢄꢅꢇꢆ; dꢇpꢆꢅꢁmꢇnꢁ ꢄf Pꢀꢆꢅmꢆꢊꢇꢈꢁiꢊꢆꢉ enginꢇꢇꢅing, sꢆngji univꢇꢅꢂiꢁy, Wꢄnjꢈ, rꢇpꢈbꢉiꢊ ꢄf Kꢄꢅꢇꢆ
ꢃ
ꢇ
ABSTRACT
ARTICLE HISTORY
Three acacetin triglycosides (compounds 1, 2 and 3) were isolated
from the herbs of Elsholtzia ciliata (Labiatae). The structure were
identified as 7-O-β-D-glucopyranosyl-(1 → 2)[α-L-rhamnopyranosyl-
rꢇꢊꢇivꢇꢃ 31 Jꢈꢉy 2017
aꢊꢊꢇpꢁꢇꢃ 2 dꢇꢊꢇmbꢇꢅ 2017
KEYWORDS
(
1 → 6)]-β-D-glucopyranoside (compound 1), 7-O-(6-O-acetyl)-β-D-
Elsholtzia ciliata; lꢆbiꢆꢁꢆꢇ;
ꢆꢊꢆꢊꢇꢁin ꢁꢅigꢉyꢊꢄꢂiꢃꢇ;
ꢆꢊꢆꢊꢇꢁin; NMr; hPlc
glucopyranosyl-(1→2)[α-L-rhamnopyranosyl-(1→6)]-β-D-glucopyranoside
compound 2) and 7-O-(6-O-acetyl)-β-D-glucopyranosyl-(1 → 2)[(4-O-
acetyl)-α-L-rhamnopyranosyl-(1 → 6)]-β-D-glucopyranoside (compound
) of acacetin. The structures of these compounds were determined on
(
3
the basis of 2D-NMR spectroscopic data. Compound 3 has not been
isolated from a natural source. In addition, the three compounds were
quantitatively analysed by HPLC. Acetylcholinesterase (AChE) inhibition
activity was assayed to find anti-Alzheimer’s activity, since this enzyme
increases the concentration of acetylcholine (ACh), a neurotransmitter,
responsible for brain’s memory. Acacetin, the aglycone of the three
compounds, exhibited a potent anti-cholinesterase activity (IC ,
5
0
5
0.33 ± 0.87), though its glycosides (1, 2 and 3) were less active. HPLC
analysis demonstrated that the three compounds were contained in
the MeOH extract in the order of compounds 2 (12.63 mg/g extract) > 3
(3.10 mg/g) > 1 (2.92 mg/g).
CONTACT hꢇꢇ-Jꢈꢀn Pꢆꢅk
ꢀjpꢆꢅk@ꢂꢆngji.ꢆꢊ.kꢅ
sꢈppꢉꢇmꢇnꢁꢆꢉ ꢃꢆꢁꢆ fꢄꢅ ꢁꢀiꢂ ꢆꢅꢁiꢊꢉꢇ ꢊꢆn bꢇ ꢆꢊꢊꢇꢂꢂꢇꢃ ꢆꢁ ꢀꢁꢁpꢂ://ꢃꢄi.ꢄꢅg/10.1080/14786419.2017.1413556.
©
2017 Infꢄꢅmꢆ uK limiꢁꢇꢃ, ꢁꢅꢆꢃing ꢆꢂ tꢆyꢉꢄꢅ & Fꢅꢆnꢊiꢂ Gꢅꢄꢈp

2
A. NUGROHO ET AL.
1
. Introduction
The genus Elsholtzia (Labiatae = Lamiaceae) consist of at least 40 species that are widely
distributed in eastern Asia and used as traditional herbs especially due to their fragrant
properties (Xue et al. 2016). Most species of this genus are rich in essential oils and fla-
vone-type compounds (Zhong et al. 2016). One of the important species is Elsholtzia ciliata,
which is used in the treatment of Alzheimer’s disease along with fever, headache, diarrhoea
and several inflammatory diseases in Korea (Kim et al. 2016). The constituents reported from
this plant are essential oil, elsholtzia ketone, flavonoids, steroids and triterpenes (Dembitskii
et al. 1993; Liu et al. 2007). Regarding the pharmacological actions, E. ciliata herb extract was
reported to inhibit renal inflammation (Kim et al. 2016) and mast cell-mediated allergy (Kim
et al. 2011). Since this plant that belongs to Labiatae contains fragrant and phenolic sub-
stances, therefore we tried to develop it as a biomaterial source with cognition-enhancing
activity.
Loss of acetylcholine concentration in brain is observed in patients with Alzheimer’s dis-
ease alongside memory impairments. Therefore, many researchers aim to develop therapeu-
tics capable of treating Alzheimer’s disease by identifying the inhibitors of acetylcholinesterase
(AChE) (Luo et al. 2016). Furthermore, it has been reported that rivastigmine, one of active
synthetic cholinesterases, is clinically effective against Alzheimer’s disease (Lai et al. 2016).
Flavonoids with anti-AChE activity have been isolated from Macaranga kurzii (Thanh et al.
2012). Many compounds possessing an α,β-unsaturated carbonyl have been documented
to show anti-AChE activity (Zha et al. 2016).
In the present study, three acacetin triglycosides were isolated from the herb of E. ciliata and
subjected to acetylcholinesterase (AChE) inhibition assay. One of the isolated compound, acacetin
7-O-(6-O-acetyl)-β-D-glucopyranosyl-(1 → 2)[(4-O-acetyl)-α-L-rhamnopyranosyl-(1 → 6)]-β-D-glu-
copyranoside (3), has not been reported previously from a natural source.
2
. Results and discussion
The UV spectra of the compounds measured with the shift reagent and the IR data of the
1
functional groups were described in the Experimental section. In the H-NMR spectrum of
compound 3, two meta-coupled peaks (J = 1.8 Hz) of H-6 and -8 were observed at δ 6.76
and δ 6.48, respectively. Two ortho-coupled (J = 8.4 Hz) assignable to H-2,6 and H-3,5 were
shown at δ 8.03 (2H) and δ 7.13 (2H), respectively. In addition, a singlet proton (1H) and a
singlet methoxyl (3H) peaks at δ 6.94 and δ 3.86 are ascribable to H-3 and 4′-OCH of the
3
aglycone. A peak of 5-OH at δ 12.9 (1H, s) indicates that free 5-OH is present in this compound.
Three anomeric protons shown at δ 5.26, δ 4.52 and δ 4.61 indicated the presence of three
sugar moieties. A doublet methyl peak shown at δ 0.92 (3H, d, J = 6.0 Hz) implied the pres-
ence of L-rhamnose. Two acetyls were also observed at δ 1.95 and 1.93.
1
Compound 3 was determined to be an acacetin triglycoside from the results of H-NMR
1
3
spectrum and the hydrolysis experiment. The C-NMR spectral data of compound 3 were
compared with the literature acacetin 7-O-β-D-glucopyranosyl-(1 → 2)[α-L-rhamnopyrano-
syl-(1 → 6)]-β-D-glucopyranoside with no acetyl group reported by Veitch et al. (2010). In the
HMBC spectrum, the anomeric proton of the inner sugar, D-glucose, was crossed to C-7
(
164.4 ppm), that of the terminal D-glucose was correlated with the C-2 (83.5 ppm) of the
inner D-glucose, and that of L-rhamnose was long-range coupled to C-6 (66.1 ppm) of

NATURAL PRODUCT RESEARCH
3
D-glucose. A considerably deshielded peak of C-2″ observed at 83.5 ppm provided an evi-
dence that a terminal sugar was attached at the position. The C-6 of D-glucose in the terminal
sugar is commonly shown in the range of 60–63 ppm. Therefore, the peak at 66.1 ppm
assigned to C-6 of the inner D-glucose is the result of sugar (L-rhamnose) attachment.
In the HMBC correlation (Figure S1), the two acetyls were confirmed to be attached to the
C -OH of the terminal D-glucose and the C -OH of L-rhamnose because the two peaks δ 1.97
6
4
and δ 1.95 were correlated with 64.0 ppm (C-6′″) and 74.4 ppm (C-4′″), respectively. The α-con-
figuration of L-rhamnose was observed from a broad singlet peak shape of the anomeric
proton shown at δ 4.61. The β-configurations of the inner- and terminal D-glucose were
determined from the J values at δ 5.26 (J = 7.2) and δ 4.52 (J = 7.8 Hz) of the anomeric proton.
Therefore, the structure of compound 3 was determined to be acacetin 7-O-(6-O-acetyl)-β-
D-glucopyranosyl-(1 → 2)[(4-O-acetyl)-α-L-rhamnopyranosyl-(1 → 6)]-β-D-glucopyranoside.
1
13
The H- and C-NMR spectral data of the three compounds were fully assigned based on
1
1
1
13
the H- H-, H- C COSY and HMBC NMR spectra (Table S1). Compound 1 with no acetyl group
was determined to have the same structure as acacetin 7-O-β-D-glucopyranosyl-(1 → 2)
[
(
α-L-rhamnopyranosyl-(1 → 6)]-β-D-glucopyranoside isolated from Robinia pseudoacacia
Veitch et al. 2010). It was also found that compound 2 possessing an acetyl has the structure
of 7-O-(6-O-acetyl)-β-D-glucopyranosyl-(1 → 2)[α-L-rhamnopyranosyl-(1 → 6)]-β-D-glucopyra-
noside as isolated from Calamintha glaudulosa (Marin et al. 2001). However, compound 3
has not been isolated from a natural source previously. Chemical structures of the three
compounds are presented in Figure 1.
The herb of E. ciliata has been used to treat Alzheimer’s disease along with headache,
fever and abdominal pain in Korea. We attempted to determine the anti-Alzheimer’s activity
by evaluating anti-AChE activity by Ellman’s method (Ellman et al. 1961). The prominent
anti-Alzheimer’s activity (IC , 50.33 μg/mL) of acacetin was shown, though the glycosides
5
0
(
1–3) are less active (Table 1). The flavonoid glycoside contained in E. ciliata may show
anti-Alzheimer’s activity through the hydrolysis.
Through the optimisation experiment, the analytical methods of the three compounds
were established in terms of linearity, sensitivity and linear range, as shown in Table 2. The
Figure 1. sꢁꢅꢈꢊꢁꢈꢅꢇ ꢄf ꢆꢊꢆꢊꢇꢁin ꢁꢅigꢉyꢊꢄꢂiꢃꢇꢂ iꢂꢄꢉꢆꢁꢇꢃ fꢅꢄm E. ciliata.

4
A. NUGROHO ET AL.
composition of the compounds calculated from the peak area based on their calibration
equations were shown in Table 3. As shown in Figure S2, compound 2 exhibited the highest
peak area among the visible peaks. The content in the extract was in the order of compounds
2
(12.63 mg/g extract) > 3 (3.10 mg/g) > 1 (2.92 mg/g). These results suggest that acacetin
glycosides may represent pharmacological actions of E. ciliata including anti-Alzheimer’s
activity.
3
. Experimental
3
.1. Instruments and reagents
Melting points were measured on an Electrothermal digital melting point apparatus and
not corrected. Optical rotations were measured on a JASCO DIP-360 digital polarimeter at
2
0°C. UV spectra were recorded on a UV-160A UV-visible recording spectrophotometer. IR
1
spectra were recorded on a JASCO 4200 FT-IR spectrometer using a KBr disc method. H- and
13
C-nuclear magnetic resonance (NMR) spectra were taken on a Bruker AM-600 spectrometer
using an internal standard tetramethylsilane (TMS). High-resolution electrospray mass spec-
tra were obtained on a Synapt G2 mass spectrometer. The ion exchange resin used for column
chromatography was Diaion HP-20 (Mitsubishi Chemical Co.). The reversed phase column
(
octadecylsilane column) used for isolation was the product of Snap cartridge (KP-C18-HS,
ODS 30 g, ϕ 3.0 × 7.5 cm). The Varian HPLC system used for the analysis consisted of Prostar
2
4
10 pumps, Prostar 325 UV–Vis detector and a Shiseido Capcell PAK C18 column (5 μm,
.6 × 250 mm, Japan).
3
.2. Plant material
E. ciliata was collected in the mountainous area of Gangwon Province in Korea. The collected
plant was cut and dried in the shade. This plant was identified by Prof. Byong-Min Song
(
(
Department of Forest Science, Sangji University, Korea) and the voucher specimen
natchem#-63) was deposited in the Laboratory of Natural Product Chemistry, Department
of Pharmaceutical Engineering, Sangji University.
3
.3. Extraction and fractionation
The plant material (2.0 kg) was extracted with 80% MeOH (20 L) under reflux for 5 h. The
extracted solution was filtered, evaporated in vacuo and further dried on a freezing dryer to
give 115 g of the 80% MeOH extract. For successive fractionation, the MeOH extract is sus-
pended in H O (1.0 L) and fractionated three times with n-hexane (each 800 mL) to give the
2
hexane fraction (26.3 g). In the same manner, chloroform (10.1 g), ethyl acetate (3.90 g) and
Table 1. Ic vꢆꢉꢈꢇꢂ ꢄf ꢁꢀꢇ ꢁꢀꢅꢇꢇ ꢆꢊꢆꢊꢇꢁin ꢁꢅigꢉyꢊꢄꢂiꢃꢇꢂ iꢂꢄꢉꢆꢁꢇꢃ fꢅꢄm Elsholtzia ciliata ꢄn acꢀe ꢆꢊꢁiviꢁy.
5
0
Compound
IC50 (μg/mL)
1
2
3
120.56 ± 3.52
156.26 ± 0.06
159.87 ± 1.65
50.33 ± 0.87
12.50 ± 0.34
3
a
apigꢇnin

NATURAL PRODUCT RESEARCH
5
Table 2. linꢇꢆꢅiꢁy ꢄf ꢂꢁꢆnꢃꢆꢅꢃ ꢊꢈꢅvꢇꢂ ꢆnꢃ ꢃꢇꢁꢇꢊꢁiꢄn/qꢈꢆnꢁifiꢊꢆꢁiꢄn ꢉimiꢁꢂ fꢄꢅ ꢁꢀꢇ ꢂꢁꢆnꢃꢆꢅꢃ ꢊꢄmpꢄꢈnꢃꢂ.
Calibration
equation (linear
Linear range
(μg/mL)
model)a
R2b
LOD (μg/mL)
c
LOQ (μg/mL)
d
Compound
tR (min)
1
2
3
10.58
13.97
17.26
y = 455.51x + 74.51
y = 242.11x + 71.16
y = 844.03x + 92.93
7.81–250.0
7.81–250.0
7.81–250.0
0.9993
0.9996
0.9992
0.28
0.53
0.13
0.92
1.77
0.42
ꢆ
y, pꢇꢆk ꢆꢅꢇꢆ ꢆꢁ 254 nm; x, ꢊꢄnꢊꢇnꢁꢅꢆꢁiꢄn ꢄf ꢁꢀꢇ ꢂꢁꢆnꢃꢆꢅꢃ (μg/ml).
b
2
R , ꢊꢄꢅꢅꢇꢉꢆꢁiꢄn ꢊꢄꢇffiꢊiꢇnꢁ fꢄꢅ ꢂix ꢃꢆꢁꢆ pꢄinꢁꢂ in ꢁꢀꢇ ꢊꢆꢉibꢅꢆꢁiꢄn ꢊꢈꢅvꢇꢂ (n = 4).
lod, ꢉimiꢁ ꢄf ꢃꢇꢁꢇꢊꢁiꢄn (S/N = 3).
ꢊ
ꢃ
loQ, ꢉimiꢁ ꢄf qꢈꢆnꢁifiꢊꢆꢁiꢄn (S/N = 10).
Table 3. cꢄnꢁꢇnꢁ ꢄf ꢆnꢆꢉyꢁꢇꢂ in ꢁꢀꢇ 80% Mꢇoh ꢇxꢁꢅꢆꢊꢁ ꢆnꢃ n-Bꢈꢁꢆnꢄꢉ fꢅꢆꢊꢁiꢄn ꢄf E. ciliata.
Compound
80% MeOH extract
n-Butanol fraction
1
2.92 (2.35)
12.63 (0.17)
3.10 (2.44)
18.65
15.01 (0.61)
80.64 (0.23)
4.58 (0.99)
100.23
2
3
tꢄꢁꢆꢉ
Nꢄꢁꢇ: tꢀꢇ ꢃꢆꢁꢆ wꢇꢅꢇ pꢅꢇꢂꢇnꢁ ꢆꢂ ꢆvꢇꢅꢆgꢇ ꢄf ꢁꢀꢅꢇꢇ ꢃꢇꢁꢇꢅminꢆꢁiꢄnꢂ. rsd vꢆꢉꢈꢇ wꢆꢂ ꢆꢃꢃꢇꢃ in ꢁꢀꢇ pꢆꢅꢇnꢁꢀꢇꢂꢇꢂ.
n-butanol (12.8 g) fractions were obtained by successive fractionation from the remaining
H O layer. The H O fraction (35.6 g) was obtained by drying the final H O layer.
2
2
2
3
.4. Isolation of compounds
The n-butanol fraction (12.0 g) was dissolved in 50 mL H O and passed through Diaion HP-20
2
column (Diaion HP-20 300 mL, 4.5 cm × 30 cm) with H O. Thereafter, 10 fractions were
2
obtained by eluting each 900 mL of 10% – 100% MeOH and concentrated: 0% MeOH fraction
(3.4 g), 10% MeOH fraction (0.65 g), 20% MeOH fraction (0.43 g), 30% MeOH fraction (0.49 g),
40% MeOH fraction (0.90 g), 50% MeOH fraction (1.10 g), 60% MeOH fraction (1.21 g), 70%
MeOH fraction (2.08 g), 80% MeOH fraction (1.94 g), 90% MeOH fraction (1.31 g), 100% MeOH
fraction (0.47 g). Each 1.0 g of the three fractions, 60, 70 and 80% MeOH fractions, were
purified on a ODS column with the eluting solvents (5.0:5.0), (5.5:4.5) and (6.0:4.0) for the
MeOH-H O solvent system to yield compounds 1 (90 mg), 2 (76 mg) and 3 (98 mg),
2
respectively.
3
.4.1. Compound 1
Pale yellowish solid. mp 205–207°C; UV λmax (log ε) (MeOH): 269.5 (4.27), 328.6 (4.30); (+AlCl3)
77.0 (4.24), 346.1 (4.30); (+AlCl + HCl) 278.0 (4.29), 339.3 (4.34); (+NaOAc) 273.6 (4.30), 328.3
2
3
−1
(
(
(
4.37); (+NaOAc + H BO ) 272.2 (4.33), 328.6 (4.38); IR ν (cm ): 3398.0 (broad, O–H), 2919.0
C-H), 1660.4 (α,β-unsaturated ketone), 1608.3, 1566.9, 1499.4, 1446.4 (aromatic C=C), 1355.7
CH ), 1262.2, 1182.2 (C–O), 1070.3, 1036.6 (glycoside C–O), 837.2, 612.3; HR-ESI-MS (positive)
3 3 max
3
+
m/z: 755.2402 [C H O + H] , calcd. 755.2398.
3
4
42 19
3
.4.2. Compound 2
Pale yellowish solid. mp 192–194°C; UV λmax (log ε) (MeOH): 269.6 (4.25), 328.5 (4.29); (+AlCl3)
77.3 (4.19), 345.4 (4.27); (+AlCl + HCl) 278.3 (4.24), 341.3 (4.33); (+NaOAc) 271.0 (4.28), 327.3
2
3

6
A. NUGROHO ET AL.
−
1
(
(
4.35); (+NaOAc + H BO ) 271.0 (4.30), 327.3 (4.35); IR ν (cm ): 3385 (broad, O–H), 2935
3 3 max
C–H), 1724.1 (ester), 1656.5 (α,β-unsaturated ketone), 1607.4, 1584.2, 1567.8, 1497.5 (aro-
matic C=C), 1369.2 (CH ), 1244.8 (C–O), 1079.0, 1033.7 (glycoside C–O)m 835.0, 604.6;
3
+
HR-ESI-MS (positive) m/z: 797.2508 [C H O + H] , calcd. 795.2503.
3
6
44 20
3
.4.3. Compound 3
Pale yellowish solid. mp 213–215°C; UV λmax (log ε) (MeOH): 269.4 (4.23), 328.8 (4.27); (+AlCl3)
76.2 (4.23), 345.5 (4.31); (+AlCl + HCl) 278.0 (4.22), 341.1 (4.32); (+NaOAc) 271.2 (4.27), 327.5
2
3
−1
(4.32); (+NaOAc + H BO ) 271.2 (4.28), 327.5 (4.31); IR ν (cm ): 3402.8 (broad, O–H), 2933.2
3 3 max
(C–H), 1733.7 (ester), 1656,6 (α,β-unsaturated ketone), 1606.4, 1584.2, 1567.4, 1497.5 (aro-
matic C=C), 1373.1 (CH ), 1244.8 (C–O), 1075.1, 1034.6 (glycoside C–O), 835.0, 605.5; HR-ESI-MS
3
+
1
(
positive) m/z: 839.2613 [C H O + H] , calcd. 839.2609. H-NMR (500 MHz, DMSO-d ) δ:
38 46 21 6
Acacetin - 6.94 (1H, s, H-3), 6.76 (1H, d, J = 1.8 Hz, H-6), 6.48 (1H, d, J = 1.8 Hz, H-8), 8.03 (1H,
d, J = 8.4 Hz, H-2′), 7.13 (1H, d, J = 8.4 Hz, H-3′), 7.13 (1H, d, J = 8.4 Hz, H-5′), 8.03 (1H, d,
J = 8.4 Hz, H-6′), 3.86 (3H, s, OCH ), Glc - 5.26 (1H, d, J = 7.2 Hz, H-1″), 3.87 (1H, m, H-6v), 3.52
3
(
1H, m, H-6″), Glc - 4.52 (1H, d, J = 7.8 Hz, H-1′″), 4.14 (1H, m, H-6′″), 4.01 (1H, m, H-6′″), 1.97
(
1
3H, s, 6′″–COCH ), Rha - 4.61 (1H, br s, H-1″″), 4.72 (1H, m, H-4″″), 0.92 (1H, d, J = 6.0 Hz, H-6″″),
3
13
.95 (3H, d, s, 4″″–COCH3); C-NMR (125 MHz, DMSO-d ) δ: Acacetin - 163.2 (C-2), 104.3 (C-3),
6
182.5 (C-4), 157.4 (C-5), 100.1 (C-6), 164.4 (C-7), 95.3 (C-8), 161.6 (C-9), 105.9 (C-10), 123.1
(
C-1′), 128.9 (C-2′), 115.2 (C-3′), 162.9 (C-4′), 115.2 (C-5′), 128.9 (C-6′), 56.1 (OCH ), Glc – 98.5
3
(C-1″), 83.5 (C-2″), 75.6 (C-3″), 69.4 (C-4″), 75.0 (C-5″), 66.1 (C-6″), Glc – 105.2 (C-1′″), 74.2 (C-2′″),
7
1
2
6.4 (C-3′″), 70.2 (C-4′″), 76.1 (C-5′″), 64.0 (C-6′″), 170.8 (6′″-COCH ), 21.0 (6′″-COCH ), Rha –
00.5 (C-1″″), 70.7 (C-2″″), 66.3 (C-3″″), 74.4 (C-4″″), 68.7 (C-5″″), 17.8 (C-6″″), 170.5 (4″″–COCH3),
1.3 (4″″–COCH3).
3 3
3
.5. Hydrolysis of compound 3
Compound 3 (60 mg) was hydrolysed in 5% H SO in MeOH-H O (1:1) for 5 h under reflux.
2
4
2
The cooled reaction mixture was neutralised with ammonium hydroxide, fractionated with
ethyl acetate and then the H O layer was concentrated in vacuo. D-glucose and L-rhamnose
2
were identified by checking TLC on the H O fraction. Concentration of the ethyl acetate-sol-
2
uble portion yielded compound no. 3a. The compound 3a was identified as acacetin on a
TLC, compared with the authentic compound.
3
.6. Measurement of AChE inhibitory activity
Inhibitory activity against AChE was measured using the spectrophotometric method
described by Ellman et al. (1961). ACh (acetylcholine) was used as substrates to assay the
inhibitions of AChE, respectively. The reaction mixture contained: 140 mL of sodium phos-
phate buffer (pH 8.0), 20 mL of test sample solution and 20 mL of AChE solution, which were
mixed and incubated for 15 min at room temperature. The reactions were initiated with the
addition of 10 mL of dithiobisnitrobenzoate (DTNB) and 10 mL of ACh, respectively. The
hydrolysis of ACh was monitored after the formation of yellow 5-thio2-nitrobenzoate anion
at 412 nm for 15 min, which resulted from the reaction of DTNB with thiocholine, released
by enzymatic hydrolysis of ACh.

NATURAL PRODUCT RESEARCH
7
Test samples and the positive control (berberine) were dissolved in 10% analytical grade
ethanol. All reactions were performed in triplicate in 96-well microplates, using VERSAmax
(
Molecular Devices, CA, U.S.A.). The percentage (%) inhibition was calculated from (E −
S)/E × 100, where E and S are the enzyme activities without and with the test sample, respec-
tively. The AChE inhibitory activity of each sample was expressed in terms of IC value (μg/
5
0
mL required to inhibit the hydrolysis of the substrate and ACh by 50%), as calculated from
the log–dose inhibition curve.
3
.7. Preparation of standard and sample solutions for HPLC analysis
Standard stock solutions (1000 μg/mL) were prepared by dissolving each standard com-
pound in MeOH, and stored 4°C or below. Working standard solutions were prepared by
serial dilution of standard stock solutions. The regression equation was determined by plot-
ting the peak area (y) vs. six concentrations (x, μg/mL). To prepare the sample solution, each
lyophilised extract was sufficiently dissolved in MeOH using ultrasonication. The solutions
were filtered using a disposable syringe filter (0.50 μm, Dismic-25JP, Advantec, Japan) prior
to injection into the HPLC system.
3
.8. HPLC analytical method
Two solvents, solvent A (H O with 0.05% acetic acid, v/v) and solvent B (CH CN with 0.05%
2
3
acetic acid, v/v) were used in this method. The linear gradient elution of the solvents was
programmed as follows: 0–35 min (15 → 65% B), 35–40 min (65% B), 40–42 min (65 → 100%
B), 42–46 min (100% B), 46–49 min (100 → 15% B) and 49–55 min (15% B). The flow rate and
column temperature was set constantly at 1.0 mL/min and 40 °C, respectively. The detection
wavelength was fixed at 254 nm.
4
. Conclusions
Three flavonoid glycosides were isolated from E. ciliata. Of the three compounds, a new
structure of an acacetin trisaccharide was determined as acacetin 7-O-(6-O-acetyl)-β-D-
glucopyranosyl-(1 → 2)[(4-O-acetyl)-α-L-rhamnopyranosyl-(1 → 6)]-β-D-glucopyranoside on
the basis of spectroscopic data. In the anti-cholinesterase inhibition activity, the three acace-
tin triglycosides were found less active, though acacetin of its aglycone was more active,
which suggests that it may be effective against Alzheimer’s disease. The content of three
compounds was quantitatively determined by HPLC.
Disclosure statement
No potential conflict of interest was reported by the authors.
Funding
This work was supported by the Sangji University Research Fund under [grant number 2016-0101].

8
A. NUGROHO ET AL.
ORCID
Agung Nugroho
Jae Sue Choi
Hee-Juhn Park
http://orcid.org/0000-0002-7236-9654
http://orcid.org/0000-0001-9034-8868
http://orcid.org/0000-0003-4328-9038
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