Flavanonol glucosides from the aerial parts of Agrimonia pilosa Ledeb. and their acetylcholinesterase inhibitory effects

Article abstract of DOI:10.1016/j.carres.2017.04.014

Two new flavanonol glucoside isomers, (2R,3S)-dihydrokaempferol 3-O-β-D-glucoside (1) and (2S,3R)-dihydrokaempferol 3-O-β-D-glucoside (2), were isolated from the aerial parts of Agrimonia pilosa Ledeb., along with eight known flavanonol glucosides (3–10).

Full text of DOI:10.1016/j.carres.2017.04.014

Accepted Manuscript
Flavanonol glucosides from the aerial parts of Agrimonia pilosa Ledeb. and their
acetylcholinesterase inhibitory effects
U. Min Seo, Duc Hung Nguyen, Bing Tian Zhao, Byung Sun Min, Mi Hee Woo
PII:
S0008-6215(17)30045-9
10.1016/j.carres.2017.04.014
CAR 7364
DOI:
Reference:
To appear in: Carbohydrate Research
Received Date: 20 January 2017
Revised Date: 10 April 2017
Accepted Date: 13 April 2017
Please cite this article as: U.M. Seo, D.H. Nguyen, B.T. Zhao, B.S. Min, M.H. Woo, Flavanonol
glucosides from the aerial parts of Agrimonia pilosa Ledeb. and their acetylcholinesterase inhibitory
effects, Carbohydrate Research (2017), doi: 10.1016/j.carres.2017.04.014.
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ACCEPTED MANUSCRIPT
Graphical abstract
Flavanonol glucosides from the aerial parts of Agrimonia pilosa Ledeb. and their
acetylcholinesterase inhibitory effects
U Min Seo, Duc Hung Nguyen, Bing Tian Zhao, Su Hui Seong, Jae Sue Choi, Jeong Ah
Kim, Byung Sun Min, and Mi Hee Woo

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Flavanonol glucosides from the aerial parts of Agrimonia pilosa Ledeb. and their
acetylcholinesterase inhibitory effects
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U Min Seoa, Duc Hung Nguyena,b, Bing Tian Zhaoa, Byung Sun Mina, and Mi Hee Wooa,*
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aCollege of Pharmacy, Catholic University of Daegu, Gyeongsan 38430, Republic of Korea
bPhutho College of Pharmacy, Viettri City, Phutho Province 290000, Vietnam
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*Corresponding author.
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E-mail address: woomh@cu.ac.kr (M.H. Woo).

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ABSTRACT
Two new flavanonol glucoside isomers, (2R,3S)-dihydrokaempferol 3-O-β-
(1) and (2S,3R)-dihydrokaempferol 3-O-β- -glucoside (2), were isolated from the aerial
D
-glucoside
D
parts of Agrimonia pilosa Ledeb., along with eight known flavanonol glucosides (3‒10).
Their structures were determined on the basis of spectroscopic analysis. In addition, these
compounds were evaluated to determine their acetylcholinesterase inhibitory activities. The
results indicated that these compounds have moderate inhibitory effects, with IC50 values
ranging from 76.59 ± 1.16 to 97.53 ± 1.64 ꢀM, except compounds 1 and 4 were inactive.
Keywords: Agrimonia pilosa, Flavanonol glucoside, Acetylcholinesterase activity

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1. Introduction
In Alzheimer’s disease (AD), cholinergic neurons are lost and the endogenous level
of acetylcholine is decreased. Drugs that penetrate the blood brain barrier and partially
inhibit AChE can increase the levels of acetylcholine, potentiate its physiological effects, and
provide symptomatic relief [1]. The most frequently prescribed anti-AD drugs are
cholinesterase inhibitors, which act by increasing ACh levels in the brain via the inhibition
of AChE. Several flavonoid compounds have been reported to be AChE inhibitors [2].
Agrimonia pilosa Ledeb. (AP) is a perennial herb in the Rosaceae family. It is mainly
distributed in Northern Asia and Eastern Europe. The roots and the aerial parts of AP also
have different uses in Traditional Chinese Medicine. Its roots have been used for hemostatic,
antimalarial, and anti-dysenteric treatment in Chinese herbal medicine for a long time [3].
Additionally, some flavonoids from AP plants showed significant acetylcholinesterase (AChE)
inhibitory activity [4].
Previous phytochemical studies have shown that the aerial parts of AP contain
polyphenols, phenolic glucosides, flavonoids [5-7], triterpenoids [8], and coumarins [3].
Flavanonol glucosides possess two chiral centers at C-2 and C-3 in their skeleton and give
four diastereomeric forms [(2S,3S), (2R,3R), (2R,3S), (2S,3R)]. Meanwhile, their two
diastereomeric forms [(2S,3S)-dihydrokaempferol 3-O-β-
D-glucoside and (2R,3R)-
dihydrokaempferol 3-O-β- -glucoside] were found from acai (Euterpe oleracea Mart.) pulp
D
[9]. Kato et al. also reported (2S,3S)-dihydrokaempferol 3-O-β-D-glucoside in the aerial
parts of AP [5].
In this study, we isolated flavanonol glucoside compounds from the ethyl acetate
fraction of the aerial parts of AP and elucidated their structures by spectroscopic data

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analyses (IR, UV, HRMS, and 1D and 2D NMR). Furthermore, these isolated compounds
were evaluated for AChE inhibitory effects.
2. Results and discussion
2.1. Structural elucidation
The isolation was performed via multiple chromatographic steps over silica gel, YMC
RP-18 column chromatography and a semi-preparative HPLC. In this paper, two new
flavanonol glucoside isomers (1 and 2) were isolated from the EtOAc fraction of the aerial
parts of AP, along with eight known flavanonol glucosides.
The structures of known compounds were identified as (2R,3R)-dihydrokaempferol 3-O-
β-
taxifolin 3-O-β-
taxifolin 3-O-β-
(2S,3R)-taxifolin 3-O-β-
[11], by comparing their NMR data with those in the literature (see Figure 1).
D
-glucoside (3) [9], (2S,3S)-dihydrokaempferol 3-O-β-
-glucoside (5) [10], (2R,3R)-taxifolin 4'-O-β-
-glucoside (7) [11], (2R,3R)-taxifolin 7-O-β-
-glucoside (9) [11], and (2S,3S)-taxifolin 3-O-β-
D
-glucoside (4) [5,9], (2R,3S)-
-glucoside (6) [10], (2R,3R)-
-glucoside (8) [12],
-glucoside (10)
D
D
D
D
D
D
Compound 1 was obtained as a yellow amorphous powder. The molecular formula of 1 was
determined as C21H22O11 by the positive mode HR-FAB-MS data at m/z 473.1057 [M+Na]+
(cald for C21H22O11Na, 473.1054). The IR spectrum of 1 showed absorptions for hydroxyl
1
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and carbonyl groups at 3324 and 1635 cm-1. The H and C NMR spectra of 1 displayed
characteristic signals for a flavanonol moiety due to two meta-coupled aromatic protons at
δH 6.41 (2H, m, H-6, 8), four AA'BB'-type aromatic ring protons at δH 7.99 (2H, d, J = 8.4
Hz, H-2', 6') and 7.20 (2H, d, J = 8.4 Hz, H-3', 5'), and two coupled oxymethine protons at
δH 5.84 (1H, br s, H-2) and 5.13 (1H, br s, H-3), and 15 carbon signals at δC 82.0 (C-2), 76.3
(C-3), 194.6 (C-4), 165.9 (C-5), 97.9 (C-6), 169.4 (C-7), 96.9 (C-8), 163.8 (C-9), 102.3 (C-
10), 127.3 (C-1'), 130.8 (C-2', 6'), 116.3 (C-3', 5') and 159.7 (C-4') (Tables 1 and Table 2).

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These data suggested that compound 1 contain a dihydrokaempferol moiety [5]. In addition,
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the H and C NMR signals of 1 also displayed a glucose group at δH 5.09 (1H, d, J = 7.7
Hz, H-1''), 4.03 (1H, br t, J = 7.6 Hz, H-2''), 3.90 (1H, br s, H-3''), 4.15 (2H, m, H-4'', 5''),
4.50 (1H, dd, J = 2.2, 11.6 Hz, H-6''a), and 4.30 (1H, dd, J = 5.8, 11.6, H-6''b), and δC 104.0
(C-1''), 75.6 (C-2''), 78.9 (C-3''), 72.1 (C-4''), 79.0 (C-5''), and 63.3 (C-6'') (Tables 1 and
Table 2). Acid hydrolysis of 1 yielded D-glucose which was identified by HPLC analysis
after conversion of sugar to thiocarbamoyl-thiazolidine derivative [13].
The J value of the anomeric proton [δH 5.09 (1H, d, J = 7.7 Hz)] suggested β-configuration
of D-glucose [14] which was supported by NOESY experiment (Figure 3). Additionally, the
1H-13C HMBC spectrum of 1 indicated the correlation of the oxymethine proton (δH 5.13) to
C-1' (δC 104.0) (Figure 2). With all of the above data, compound 1 was determined as
dihydrokaempferol 3-O-β-
β- -glucoside include four forms [(2R,3S), (2S,3R), (2R,3R), and (2S, 3S)]. Meanwhile,
compounds 3 and 4 were determined as (2R,3R) dihydrokaempferol 3-O-β- -glucoside and
(2S,3S) dihydrokaempferol 3-O-β- -glucoside by comparing spectroscopic data with
D-glucoside. The diastereomeric forms of dihydrokaempferol 3-O-
D
D
D
literature values, respectively [9]. The mixture of 1, 2, 3, and 4 was analyzed by high
performance liquid chromatography (HPLC) on a C18 column. The result displayed four
peaks at different retention times at 27.4, 33.1, 41.3, and 43.0 min for 3, 4, 1, and 2,
respectively. These above data suggested that compounds 1 and 2 were (2R,3S) or (2S,3R)
form (see Supplementary Material). In CD spectrum, compound 1 displayed the negative
Cotton effect at 294 nm and positive one at 341 nm. The CD spectrum of 1 was similar to
those of (2R,3S)-cis-dihydrokaempferol reported (Figure 4) [15], suggesting that the
stereochemistry of 1 was (2R,3S). Therefore, compound 1 was determined as (2R,3S)-
dihydrokaempferol 3-O-β-D-glucoside, a new natural product.

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Compound 2 was obtained as a yellow amorphous powder. The molecular formula of 2
was determined as C21H22O11 by the positive mode HR-FAB-MS data at m/z 473.1058
1
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[M+Na]+ (cald for C21H22O11Na, 473.1054). The H and C NMR spectra of 2 were similar
to those of 1 except for J values of H-2 and H-3 [(br s) in 1 and (d, J = 2.8 Hz) in 2]. In 1H-
13C HMBC spectrum, compound 2 displayed the correlation between H-3 [δH 5.29 (d, J = 2.8
Hz)] and C-1'' (δC 104.8) (Figure 2). These data suggested that 2 also was a
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dihydrokaempferol 3-O-glucoside. The sugar moiety was identified as β-D-glucoside by
methods mentioned above and J value of the anomeric proton [δH 5.39 (d, J = 7.7 Hz)]. The
correlation of H-2 to H-3 was also observed in NOESY experiment. In CD experiment,
compound 2 displayed the positive Cotton effect at 296 nm and the negative one at 333 nm
that was opposite to those of 1, suggesting that the stereochemistry of 2 was (2S,3R). The CD
spectrum of 3 was positive at 291 nm and the negative Cotton effect at 328 nm, indicating
that the stereochemistry of 3 was (2R,3R) [15]. Similarly, CD spectrum of 4 showed the
negative Cotton effect at 294 nm and the positive one at 323 nm and, suggesting that the
stereochemistry of 4 was (2S,3S) (Figure 4) [15]. Finally, compound 2 was determined to be
(2S,3R)-dihydrokaempferol 3-O-β-D-glucoside, a new natural product.
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2.2. Acetylcholinesterase activity
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AChE is targeted by cholinesterase inhibitors employed for the treatment of senile dementia,
myasthenia, gravis, Parkinson’s disease, and ataxia. The AChE inhibitory effects of isolated
compounds were measured using the spectrophotometer method developed by Ellman [16].
Dehydroevodiamine was used as the positive control [4]. Compounds 1‒10 were evaluated
for inhibitory AChE effects at the applied concentrations (4, 20, and 100 ꢀM). The results
indicated that these compounds have moderate inhibitory activity, with IC50 values ranging

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from 76.59 ± 1.16 to 97.53 ± 1.64 ꢀM. Compounds 1 and 4 were very weak or inactive (IC50
values > 100 ꢀM). Some conclusions were deduced from investigating the structure-activity
relationships of these flavanonol glucosides. Compound 2, a (2S,3R) form of
dihydrokaempferol 3-O-β-
Compounds 6, 7, and 8, the diastereomeric (2R,3R) forms of taxifolin-glucoside, displayed
similar inhibitory effects on the AChE enzyme. This result indicates that the position of β-
D-glucoside, displayed better inhibitory effect than the other forms.
D
-
glucose at C-4', C-3, and C-7 in the structure of taxifolin glucoside did not affect the
inhibitory effects of the compounds. AChE inhibitor is used as a drug for the symptomatic
treatment of Alzheimer’s disease [17]. A survey of medicinal plants and their extracts to
evaluate their medicinal and therapeutic potential revealed that plants rich in polyphenols and
flavonoids have the potential to act as AChE inhibitors [17,18]. The flavanonol glucoside
compounds in this study may have partial benefits for the treatment of Alzheimer’s disease.
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3. Conclusion
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Flavonoids are a huge class in nature and their biological activities are known as antiviral,
antitumor, antiplatelet, anti-inflammatory, and antibacterial [19]. Previous studies showed
that flavonoid constituents in AP possess potential inhibitory PTP1B and AChE activities
[4,19]. In our study, two new flavanonol glucosides (1 and 2) were isolated from aerial parts
of AP, along with eight known flavanonol glucosides (3‒10). These compounds were
investigated for AChE inhibitory activity. Most of the isolates displayed moderate inhibitory
activity, except for inactive compounds 1 and 4.
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4. Experimental
4.1. General experimental procedures

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The specific rotations were determined on a JASCO DIP-370 digital polarimeter. The
circular dichroism (CD) spectra were recorded in methanol on the JASCO J–715
spectropolarimeter (Jasco, Easton, MD, USA). The infrared (IR) spectra were obtained on a
Mattson Polaris FT/IR-300E spectrophotometer. UV spectra were measured in MeOH using a
Shimadzu spectrophotometer. The nuclear magnetic resonance (NMR) spectra were recorded
in pyridine-d5 or methanol-d4 on an Oxford AS 400 MHz instrument (Varian, Palo Alto, CA,
USA) or a Bruker 500 MHz instrument (Bruker, Billerica, MA, USA). Mass spectra were
recorded using a Quattro II mass spectrometer. Column chromatography was performed
using silica gel (Merck, 63–200 µm particle size), RP-18 (Merck, 150 µm particle size), and
Sephadex LH-20 (Pharmacia Co. Ltd.). Thin Layer Chromatography (TLC) tests were
performed on silica gel 60 F254 plates (Merck, Darmstadt, Germany). Fractions were
monitored by TLC and spots were visualized by spraying with 10% H2SO4 in ethanol,
followed by heating. The semi-preparative HPLC runs were carried out using a Gilson system
with an UV detector and an Optima Pak C-18 column (10 × 250 mm, 10 µm particle size, RS
Tech. Corp., Korea). The analyses were conducted on an HPLC chromatography (Waters,
Texas, USA) and a Kinetex C-18 column (4.6 × 250 mm, 5 µm particle size, Phenomenex.,
USA). All other chemicals and solvents were of analytical grade and used without further
purification.
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4.2. Plant material
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The aerial parts of AP were collected from the herbal garden at the Catholic University
of Daegu, Korea, in August 2012. The plant material was authenticated by one of the authors
(B. S. Min). A voucher specimen (AP-2012143) has been deposited at the College of
Pharmacy, Catholic University of Daegu, Republic of Korea.

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4.3. Extraction and isolation
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The aerial parts of Agrimonia pilosa Ledeb. (32 Kg) were initially dried (5.8 Kg). Then it
was extracted with 80% EtOH (3 × 4 L) at room temperature for 7 days. The ethanol extract
was then concentrated under reduced pressure (2.0 Kg). The ethanol extract was further
suspended in H2O (1.5 L) and successfully partitioned with CH2Cl2 (1 L × 7, 800 g), EtOAc
(1 L × 7, 163.9 g), n-BuOH (1 L × 5, 500.5 g) and H2O-soluble fraction. The EtOAc fraction
(163.9 g) was subjected to open flash column chromatography over silica gel, eluted with
CH2Cl2/MeOH (100:0, 50:1, 25:1, 20:1, 15:1, 12:1, 10:1, 9:1, 8:1, 7:1, 6:1, 5:1, 4:1, 3:1, 2:1,
and 1:1) to give 19 fractions (A‒S). Fraction O (4.1 g) was then subjected to RP-C18 column
chromatography, eluted with MeOH/H2O (1:2, 2:1, 4:1, 8:1, and 10:1) to afford six
subfractions (OA‒OF). Sub-fraction EA-O-A (820 mg) was chromatographed on a Sephadex
LH-20 column and eluted with MeOH/H2O (1:1) to yield compounds 6 (8.3 mg) and
compound 8 (5.2 mg), respectively. Sub-fraction OA1 (133 mg) was subjected to semi-
preparative Gilson HPLC [YMC C18 column (10 × 250 mm, 5 ꢀm particle size)] eluted with
ACN/H2O (18:82, flow rate 2 mL/min, UV 254 nm) to yield compounds 3 (tR 27.4 min, 6.2
mg), 4 (tR 33.1, min 5.1 mg), 1 (tR 41.3 min, 3.4 mg), and 2 (tR 43.0 min, 4.1 mg). Sub-
fraction OB (1.1 g) was further purified by semi-preparative HPLC [YMC C18 column (10 ×
250 mm, 5 ꢀm particle size)] eluted with MeOH/H2O (33:67, flow rate 2 ml/min, UV 254 nm)
to yield compounds 7 (tR 26.5 min, 18.9 mg), 10 (tR 28.1 min, 8.7 mg), 5 (tR 53.0 min, 4.3
mg), and 9 (tR 55.2 min, 2.9 mg), respectively.
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4.3.1. (2R,3S)-dihydrokaempferol 3-O-β-D-glucoside (1)

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Yellow amorphous powder; [ꢀ]ꢁꢂꢃ ‒71 (c, 0.2, MeOH); UV (MeOH) λmax nm 214, 296;
1
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IR (KBr) νmax cm-1 : 3324, 2943, 2831, 1635, 1448, 1115, 1024; For H and C NMR data
see Tables 1 and 2; HR-FAB-MS m/z 473.1057 [M+Na]+ (calcd for C21H22O11Na, 473.1054).
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4.3.2. (2S,3R)-dihydrokaempferol 3-O-β-D-glucoside (2)
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Yellow amorphous powder; [ꢀ]ꢁꢂꢃ +20 (c, 0.2, MeOH); UV (MeOH) λmax nm 214, 296 ;
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IR (KBr) νmax cm-1 : 3329, 2943, 2831, 1634, 1449, 1112, 1024. For H and C NMR data
see Tables 1 and 2; HR-FAB-MS m/z 473.1058 [M+Na]+ (calcd for C21H22O11Na, 473.1054).
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4.3.3. Acid hydrolysis and HPLC analysis for 1 and 2
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Each compound (2 mg) was dissolved in 2 mol/L HCl (10 mL) and then heated at 80 ºC
for 6 h. The mixtures were concentrated in vacuo to dryness, and the residue was partitioned
between CH2Cl2 and H2O. The aqueous layer was evaporated to dryness. The dried product
was then dissolved in anhydrous pyridine (1.0 mL) and D-cysteine methyl ester hydrochloride
(4.0 mg) and the mixture was further heated at 60 ºC for 1 h. After the reaction mixture was
concentrated to dryness, o-tolyl isothiocyanate (10 ꢀL) was added, and the mixture was
heated at 60 ºC for 1 h. The product of the reaction mixture was dissolved in 1 mL MeOH
and analyzed using HPLC chromatography (Waters, Houston, TX, USA) with a Kinetex C18
column (4.6 × 250 mm, 5 ꢀm particle size; Phonomenex, Torrance, CA, USA).
Chromatographic analytical methods utilized MeOH containing 0.05% H3PO4 in H2O (20%
MeOH to 35% MeOH) for 40 min at a flow rate of 0.8 mL/min, with ultraviolet (UV)
detection at 227 nm. The reaction conditions for authentic samples were the same as
described above. Comparison of the retention times for β-D-glucose using 1 and 2 in aqueous

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solution was carried out (tR 27.66 and 27.45 min of 1 and 2, respectively) using authentic
samples (tR 27.45 min of β-D-glucose) (Supplementary material).
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4.3.4. Acetylcholinesterase inhibitory assay
Ellman’s method was applied for evaluating AChE inhibitory activity of isolated
210 compounds (Ellman et al., 1961). Acetylthiocholine iodide (ATCI) was used as substrates.
211 Briefly, the mixtures of 140 ꢀL of sodium phosphate buffer (pH 8.0), 20 ꢀL of each tested
212 compound with different concentrations (4, 20, and 100 ꢀM), and 20 ꢀL enzyme solution were
213 incubated at room temperature for 15 min. The reactions were initiated by the addition of 10
214 ꢀL of 0.01 M 5,5-bisdithionitrobenzoic acid (DTNB) and 10 ꢀL of 0.075 ATCI. The reaction
215 solutions were then incubated at 37 ºC for 20 min. A yellow compound (5-thio-nitrobenzoate)
216 production was produced by the reaction of the product thiocholine with DTNB. It was
217 detected at 412 nm. All tested samples and the positive control (dehydroevodiamine) were
218 dissolved in 10% analytical grade dimethyl sulfoxide (DMSO). The reaction was performed in
219 triplicate and recorded in 96-well microplates using a microplate reader [VERSA max
220 (Molecular Devices, Sunnyvale, CA, USA)]. Percent inhibition was calculated using the
221 formula: % AChE inhibition = (1-S/E) × 100, where E and S were the enzyme activities with
222 and without the tested sample, respectively. The 50% inhibition concentration of each tested
223 compound was calculated from the log dose-inhibition the log dose-inhibition curve.
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Acknowledgements
This work was supported by research grants from Catholic University of Daegu in 2017.
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Supplementary data
Supplementary data associated with this article can be found, in the online version.

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[19] R. Vinayagam, B. Xu, Nutr. Metab. 12 (2015) 1‒20.

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255
256
Figure captions
Figure 1. Chemical structures of compounds 1‒10 from the aerial parts of A. pilosa Ledeb.
257
258
259
Figure 2. Key HMBC (
Figure 3. Key NOESY (
) correlations established for compounds 1 and 2.
) correlations established for compounds 1 and 2.
Figure 4. CD spectra of compounds 1–4.
260

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261
Figure 1. .
R3
R2
R4
OH
2'
2'
4'
5'
4'
5'
8
8
R2
O
O
2
HO
O
O
2
9
R
S
9
6'
6'
3
10
3
10
6
6
R1
R1
OH
OH
262
Compound
R1
R2
R3
H
H
R4
Compound
R1
R2
H
H
OH
OH
α-O-Glc
β-O-Glc
α-O-Glc
β-OH
β-O-Glc
β-OH
OH
OH
OH
OH
OH
OH
OH
OH
β-O-Glc
α-O-Glc
β-O-Glc
α-OH
1
3
5
6
7
8
2
4
9
OH
OH O-Glc
OH
10
OH
OH
O-Glc OH
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264
265

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266
Figure 2
267
268

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269
Figure 3.
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271
272

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273
Figure 4.
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275

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276
Table 1
277 1H NMR data of compounds 1, 2, 3, and 4 in pyridine-d5 (500 MHz)
1
2
3
4
No
δH
δH
δH
δH
2
3
5.84 (br s)
5.13 (br, s)
5.82 (d, 2.8)
5.29 (d, 2.8)
5.78 (d, 7.6)
5.35 (d, 7.6)
5.84 (d, 8.3)
5.44 (d, 8.3)
4
5
6
6.41 (m)
6.41 (m)
6.39 (m)
6.39 (m)
6.37 (d, 1.7)
6.42 (d, 1.7)
6.35 (1.9)
7
8
6.44 (d, 1.9)
9
10
1'
2'
3'
4'
5'
6'
1''
2''
3''
4''
5''
7.99 (d, 8.4)
7.20 (d, 8.4)
7.79 (d, 8.3)
7.11 (d, 8.3)
7.64 (d, 8.4)
7.17 (d, 8.4)
7.71 (d, 8.5)
7.21 (d, 8.5)
7.20 (d, 8.4)
7.99 (d, 8.4)
5.09 (d, 7.7)
4.03 (br t, 7.6)
3.90 (br s)
4.15 (m)
7.11 (d, 8.3)
7.79 (d, 8.3)
5.39 (d, 7.7)
4.05 (br t, 8.3)
3.97 (br, s)
7.17 (d, 8.4)
7.64 (d, 8.4)
5.49 (d, 7.8)
4.01 (m)
7.21 (d, 8.5)
7.71 (d, 8.5)
4.93 (d, 7.4)
4.14 (m)
4.01 (m)
3.85 (m)
4.13 (br t, 9.2)
4.26 (m)
4.14 (br t, 9.2)
4.31 (m)
4.14 (m)
4.15 (m)
4.21 (m)
4.50 (dd, 2.2,
11.6)
4.54 (dd, 2.1,
11.6)
4.53 (dd, 2.1,
11.8)
6''a
6''b
4.47 (br d, 11.4)
4.26 (m)
4.30 (dd, 5.8,
11.6)
4.35 (dd, 2.1,
11.8)
4.31 (m)
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280 Table 2
281 13C NMR data of compounds 1, 2, 3, and 4 in pyridine-d5 (125 MHz)
1
2
3
4
No.
δC
δC
δC
δC
2
3
82.0
81.7
83.2
83.0
76.3
78.4
77.2
76.8
4
194.6
165.9
97.9
194.7
165.8
97.9
195.2
165.8
97.9
194.9
165.7
97.8
5
6
7
169.4
96.9
169.2
96.8
169.8
96.8
169.6
96.7
8
9
163.8
102.3
127.3
130.8
116.3
159.7
116.3
130.8
104.0
75.6
163.9
102.6
127.3
130.9
116.6
159.8
116.6
130.9
104.8
75.5
163.6
102.5
127.6
96.9
163.6
102.8
127.9
130.4
116.9
160.1
116.9
130.4
103.3
76.8
10
1'
2'
3'
4'
5'
6'
1''
2''
3''
4''
5''
6''
116.7
160.0
116.7
130.4
105.4
76.0
78.9
78.9
79.1
79.4
72.1
72.2
72.0
75.5
79.0
78.9
78.8
78.7
63.3
63.5
63.5
63.2
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283

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285
Table 3
AChE inhibitory activity of compounds 1‒10.
Compounds
IC50 valuea (ꢀM)
> 100
1
76.59 ± 1.16
89.29 ± 3.80
> 100
2
3
4
97.53 ± 1.64
79.00 ± 0.05
78.27 ± 1.47
78.59 ± 0.93
96.29 ± 2.93
91.35 ± 2.44
34.05 ± 1.11
5
6
7
8
9
10
Dehydroevodiamineb
286
287
aThe values indicate 50% AChE inhibitory effect. These data represent the average values of
three repeated experiments.
288
Dehydroevodiamineb was used as a positive control.

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Research highlights
Flavanonol glucosides from the aerial parts of Agrimonia pilosa Ledeb. and their
acetylcholinesterase inhibitory effects
U Min Seo, Duc Hung Nguyen, Bing Tian Zhao, Su Hui Seong, Jae Sue Choi, Jeong Ah
Kim, Byung Sun Min, and Mi Hee Woo
ꢀ Two new flavanonol glucoside isomers and eight known compounds were isolated
from Agrimonia pilosa Ledeb.
ꢀ Their structures were determined on the basis of spectroscopic analysis
ꢀ All isolated compounds were evaluated for their AChE inhibitory activities
ꢀ Most of isolates showed moderate inhibitory effects