Two new flavonoids from Artemisia argyi with their anticoagulation activities

Article abstract of DOI:10.1080/14786419.2017.1332603

A new flavone glycoside, eupatilin 7-O-β-d-glucopyranoside (1) and a new flavone, 5,6,2′,4′-tetrahydroxy-7,5′-dimethoxyflavone (2), were isolated from Artemisia argyi. Their structures were unambiguously elucidated by extensive spectroscopic analysis. Both flavonoids were evaluated for in vitro anticoagulation activities. Compound 1 significantly extended thrombin time. Compound 2 had obvious effect in increasing prothrombin time.

Full text of DOI:10.1080/14786419.2017.1332603

Natural Product Research
Formerly Natural Product Letters
ISSN: 1478-6419 (Print) 1478-6427 (Online) Journal homepage: http://www.tandfonline.com/loi/gnpl20
Two new flavonoids from Artemisia argyi with
their anticoagulation activities
Jie-Li Lv, Zhen-Zhen Li & Lai-Bin Zhang
To cite this article: Jie-Li Lv, Zhen-Zhen Li & Lai-Bin Zhang (2017): Two new flavonoids
from Artemisia argyi with their anticoagulation activities, Natural Product Research, DOI:
10.1080/14786419.2017.1332603
To link to this article: http://dx.doi.org/10.1080/14786419.2017.1332603
View supplementary material
Published online: 25 May 2017.
Submit your article to this journal
View related articles
View Crossmark data
Full Terms & Conditions of access and use can be found at
http://www.tandfonline.com/action/journalInformation?journalCode=gnpl20
Download by: [The UC San Diego Library]
Date: 26 May 2017, At: 15:13

Natural Product research, 2017
ꢁꢁpꢂ://ꢃꢄi.ꢄꢅg/10.1080/14786419.2017.1332603
ꢀ
Two new flavonoids from Artemisia argyi with their
anticoagulation activities
†
†
Jie-Li Lv , Zhen-Zhen Li and Lai-Bin Zhang
sꢆꢀꢄꢄꢇ ꢄf Pꢀꢈꢅmꢈꢆy, Xinxiꢈng Mꢉꢃiꢆꢈꢇ univꢉꢅꢂiꢁy, Xinxiꢈng, Pꢉꢄpꢇꢉ’ꢂ rꢉpꢊbꢇiꢆ ꢄf cꢀinꢈ
ABSTRACT
ARTICLE HISTORY
A new flavone glycoside, eupatilin 7-O-β-d-glucopyranoside (1) and
a new flavone, 5,6,2′,4′-tetrahydroxy-7,5′-dimethoxyflavone (2), were
isolated from Artemisia argyi. Their structures were unambiguously
elucidated by extensive spectroscopic analysis. Both flavonoids
were evaluated for in vitro anticoagulation activities. Compound 1
significantly extended thrombin time. Compound 2 had obvious
effect in increasing prothrombin time.
rꢉꢆꢉivꢉꢃ 15 dꢉꢆꢉmbꢉꢅ 2016
aꢆꢆꢉpꢁꢉꢃ 8 Mꢈy 2017
KEYWORDS
Artemisia argyi; cꢄmpꢄꢂiꢁꢈꢉ;
flꢈvꢄnꢉ; ꢈnꢁiꢆꢄꢈgꢊꢇꢈꢁiꢄn
ꢈꢆꢁiviꢁy
Artemisia argyi
1
. Introduction
Thromboembolic diseases continue to be the leading cause of death throughout the world
Wu et al. 2015). As is well-known, thrombosis is closely related to activating platelet adhe-
(
sion, aggregation, secretion functions and activation of intrinsic and extrinsic coagulation
systems, which cause blood coagulation and fibrin formation (Xin et al. 2011). Therefore,
anticoagulants play a pivotal role in the prevention and treatment of thrombotic disorders
(
Xu et al. 2016).
The genus Artemisia (Compositae) consists of about 500 species and subspecies mainly
distributed in the temperate zones of North America, Europe and Asia. Sesquiterpenoids
and flavonoids were the most abundant secondary metabolites in Artemisia species (Ornano
et al. 2016; Allison et al. 2017; Peron et al. 2017). Artemisia argyi is one of the most widely
distributed Artemisia species in China. The dried leaves of A. argyi, known as‘Aiye ’, was firstly
recorded as medicine in ‘Ming Yi Bie Lu’ in the Liang Dynasty (A.D. 502–560). A. argyi leaf
CONTACT lꢈi-Bin Zꢀꢈng
tꢀꢉꢂꢉ ꢈꢊꢁꢀꢄꢅꢂ ꢆꢄnꢁꢅibꢊꢁꢉꢃ ꢉqꢊꢈꢇꢇy ꢁꢄ ꢁꢀꢉ wꢄꢅk.
zꢀꢈngꢇb@xxmꢊ.ꢉꢃꢊ.ꢆn
†
sꢊppꢇꢉmꢉnꢁꢈꢇ ꢃꢈꢁꢈ fꢄꢅ ꢁꢀiꢂ ꢈꢅꢁiꢆꢇꢉ ꢆꢈn bꢉ ꢈꢆꢆꢉꢂꢂꢉꢃ ꢈꢁ ꢀꢁꢁpꢂ://ꢃꢄi.ꢄꢅg/10.1080/14786419.2017.1332603.
©
2017 Infꢄꢅmꢈ uK limiꢁꢉꢃ, ꢁꢅꢈꢃing ꢈꢂ tꢈyꢇꢄꢅ & Fꢅꢈnꢆiꢂ Gꢅꢄꢊp

2
J.ꢀL. LV ET AL.
Figure 1. sꢁꢅꢊꢆꢁꢊꢅꢉꢂ ꢄf ꢆꢄmpꢄꢊnꢃꢂ 1 ꢈnꢃ 2.
from Qichun, Hubei province (Qi Zhou, in ancient times), is traditionally regarded as the
genuine Aiye (Lv et al. 2013; Ge et al. 2016). It has been used as a traditional Chinese herb
for moxibustion and for curing homeostasis, menstruation-related symptoms, eczema, diar-
rhoea and tuberculosis (Wang et al. 2013). Phytochemical researches on this species have
led to the isolation of sesquiterpenoids, triterpenoids, steroids, coumarins and flavonoids,
showing anti-inflammatory, immunomodulatory, antitumour, antimutagen and antimicro-
bial activities (Nakasugi et al. 2000; Lee et al. 2002, 2005; Liu et al. 2006; Ji et al. 2010; Wang
et al. 2013; Zhang et al. 2013; Wang et al. 2014; Zeng et al. 2014). Particularly, the essential
oil from A. argyi has obvious effect on improving haemorheology in the rat acute blood stasis
model (Ge et al. 2016). 5,7-Dihydroxy-6,3′,4′-trimethoxy-flavone from the leaves of A. argyi
exhibited antiplatelet aggregation activity in vitro (Zhong & Cui 1992). In further research
on the anticoagulation-active constituents of A. argyi, two new flavonoids (1 and 2)
(
Figure 1) were isolated and characterised, respectively. Herein, the isolation and structural
elucidation of the new compounds and their anticoagulation activities are described.
2
. Results and discussion
2
.1. Structure elucidation of two new flavonoids
Compound 1 was isolated as a yellowish amorphous powder. Its molecular formula was
+
established as C H O on the basis of the HRESI-MS ion at m/z 529.1313 [M + Na] (Calcd
24
26 12
for C H O Na, 529.1322), indicating 12° of unsaturation. The UV maximum absorptions at
2
4
26 12
275 and 338 nm of 1 were characteristic of a flavone skeleton (Ngankeu Pagning et al. 2016;
Xiao et al. 2016), which was corroborated by the absorption bands at 3380 (hydroxyl), 1662
−1
13
(
conjugated carbonyl) and 1614 and 1465 (aromatic ring) cm in its IR spectrum. In the C
NMR and HSQC spectra, 24 carbon signals were exhibited, corresponding to a flavone skel-
eton with one glucosyl unit and three methoxy groups.
1
In the H NMR spectrum of 1, the appearance of a typical ABX system at δ 7.71 (1H, dd,
H
J = 8.6, 1.4 Hz, H-6′), 7.59 (1H, d, J = 1.4 Hz, H-2′) and 7.15 (1H, d, J = 8.6 Hz, H-5′) corresponded
to a 1′,3′,4′-trisubstituted phenyl moiety of ring B, which was further confirmed through
HMBC correlations (Figure S11) from H-2′ to C-2, C-3′, C-4′ and C-6′, from H-5′ to C-1′, C-3′
and C-4′ and from H-6′ to C-2, C-2′ and C-4′. In addition, the other two aromatic protons were
observed on this spectrum, assignable to H-8 (δ 7.08, 1H, s) and H-3 (δ 7.07, 1H, s) by
H
H
analyses of the HMBC correlations from H-8 to C-6, C-7, C-9 and C-10 and from H-3 to C-2,
1
C-4, C-10 and C-1′. The H NMR spectrum of 1 also exhibited a hydrogen-bonded hydroxyl
proton signal at δ 12.92 (1H, s, OH-5) that can be assigned to the OH-5 group, as further
H

NATURAL PRODUCT RESEARCH
3
confirmed on the basis of the correlations between δ 12.92 (OH-5) and δ 152.3 (C-5), 132.6
H
C
(
C-6) and 105.8 (C-10) in the HMBC spectrum. Moreover, three methoxy singlets at δ 3.78,
H
3.86 and 3.88 (each 3H, s) were observed. The locations of the three methoxy groups at C-6,
C-4′ and C-3′ were inferred from the HMBC correlations between the methoxy protons and
1
C-6/C-4′/C-3′, respectively. This H-NMR spectrum also showed the presence of glycosyl unit
with the anomeric proton at δ 5.13 (1H, overlapped, H-1″), a methylene group at δ 3.74
H
H
(
1H, m, H -6″) and 3.50 (1H, overlapped, H -6″). Other protons (H-2″-H-5″) of the sugar moiety
a
b
were observed between 3.19 and 3.47 ppm. The signals at δ 5.48 (1H, d, J = 4.9 Hz), 5.19
H
(
1H, d, J = 4.3 Hz), 5.13 (1H, overlapped) and 4.68 (1H, t, J = 5.2 Hz) were assigned to OH-2″,
OH-3″, OH-4″ and OH-6″, respectively, as supported by the HMBC correlations of OH-2″/C-2″,
C-1″, C-3″; OH-3″/C-3″, C-2″, C-4″; OH-4″/C-4″, C-3″, C-5″ and OH-6″/C-6″, C-5″.
Acid hydrolysis of 1 followed by TLC analysis and optical rotation of the hydrolysate and
direct comparison with authentic sugars further indicated the presence of a d-glucose unit.
Simultaneously, the anomeric configuration of glucosyl moiety was assigned as β on the
basis of the chemical shift of its anomeric carbon (δ 100.4). The glucosyl residue was located
C
at C-7 of the aglycon flavone by the appearance of HMBC cross peak of H-1″ (δ 5.13) with
H
C-7 (δ 156.6). Therefore, the structure of compound 1 was elucidated as 5-hydroxy-6,3′,4′-tri-
C
methoxyflavone 7-O-β-d-glucopyranoside, named eupatilin 7-O-β-d-glucopyranoside.
Compound 2 was obtained as a yellow powder. The molecular formula of compound 2
was determined to be C H O by HRESI-MS, consistent with the molecular ion peak at m/z
1
7
14
8
+
3
69.0584 [M + Na] (Calcd for C H O Na, 369.0586). The UV spectrum of 2 showed absorp-
17 14 8
tion bands at λmax 283 and 367 nm, which were characteristic of a flavone skeleton. The IR
−1
signals at 3375, 1663, 1603 and 1428 cm suggested the presence of hydroxyl, carbonyl
1
3
and aromatic functionalities. The C NMR and HSQC spectra exhibited 17 carbon signals,
comprising a flavone skeleton with two methoxy groups.
1
The H NMR spectrum of 2 exhibited a characteristic proton signal at δ 12.78 correspond-
H
ing to a chelated hydroxyl group at C-5, which was confirmed by the HMBC correlations
(Figure S11) from OH-5 to C-5, C-6 and C-10. Moreover, four aromatic protons were observed
on this spectrum, assignable to H-3 (δ 7.08, 1H, s), H-8 (δ 6.94, 1H, s), H-3′ (δ 6.57, 1H, s)
H
H
H
and H-6′ (δ 7.45, 1H, s) by analyses of the HMBC correlations from H-3 to C-2, C-4, C-10 and
H
C-1′, from H-8 to C-6, C-7, C-9 and C-10, from H-3′ to C-1′, C-2′, C-4′ and C-5′ and from H-6′
1
to C-2, C-1′, C-2′, C-4′ and C-5′. Additionally, the other H NMR spectral information include
two methoxy groups (δ 3.93 and 3.82), three hydroxyl groups at δ 8.65 (1H, br s, OH-6),
H
H
10.42 (1H, br s, OH-2′) and 10.03 (1H, br s, OH-4′). The long-range HMBC correlations from
one methoxy group at δ 3.93 (OCH -7) to C-7 (δ 154.2) and from another one at δ 3.82
H
3
C
H
(
OCH -5′) to C-5′ (δ 141.6) revealed that they were located at C-7 and C-5′, respectively.
3 C
Furthermore, the HMBC correlations from OH-6 to C-6/C-5/C-7, from OH-2′ to C-2′/C-1′/C-3′
and from OH-4′ to C-4′/C-3′/C-5′ confirmed that the three hydroxyl groups were attached
to C-6, C-2′ and C-4′, respectively. Therefore, the structure of compound 2 was determined
as 5,6,2′,4′-tetrahydroxy-7,5′-dimethoxyflavone.
2
.2. Anticoagulation activities
Compounds 1 and 2 were evaluated for their in vitro anticoagulation activities on the throm-
bin time (TT) and prothrombin time (PT). As shown in Figure 2, compound 1 remarkably
prolonged the TT with a good dose–effect relationship at the concentration from 20 to

4
J.ꢀL. LV ET AL.
Figure 2. anꢁiꢆꢄꢈgꢊꢇꢈnꢁ ꢈꢆꢁiviꢁiꢉꢂ ꢄf ꢆꢄmpꢄꢊnꢃ 1 (c1) ꢈnꢃ ꢆꢄmpꢄꢊnꢃ 2 (c2) ꢈꢁ ꢃiffꢉꢅꢉnꢁ ꢆꢄnꢆꢉnꢁꢅꢈꢁiꢄnꢂ
wiꢁꢀ ꢅꢉꢂpꢉꢆꢁ ꢁꢄ tt ꢈnꢃ Pt in vitro. tꢀꢉ ꢈꢆꢁiviꢁy iꢂ ꢉxpꢅꢉꢂꢂꢉꢃ ꢈꢂ ꢁꢀꢉ ꢆꢇꢄꢁꢁing ꢁimꢉ in ꢂꢉꢆꢄnꢃꢂ (ꢂ). dMso wꢈꢂ
ꢊꢂꢉꢃ ꢈꢂ ꢁꢀꢉ ꢆꢄnꢁꢅꢄꢇ. hꢉpꢈꢅin ꢂꢄꢃiꢊm (0.5 μM) wꢈꢂ ꢊꢂꢉꢃ ꢈꢂ ꢁꢀꢉ pꢄꢂiꢁivꢉ ꢆꢄnꢁꢅꢄꢇ. Vꢈꢇꢊꢉꢂ ꢈꢅꢉ ꢉxpꢅꢉꢂꢂꢉꢃ ꢈꢂ
*
**
***
mꢉꢈnꢂ ± sd (n = 3) ꢈnꢃ p < 0.05, p < 0.01 ꢈnꢃ p < 0.001 ꢈꢂ ꢆꢄmpꢈꢅꢉꢃ wiꢁꢀ ꢁꢀꢉ ꢆꢄnꢁꢅꢄꢇ gꢅꢄꢊp.
50 mM, compared with the DMSO control. However, 2 had significant effect in increasing
PT at the concentration from 40 to 50 mM.
PT value reflects the activity of the extrinsic pathways of coagulation. TT is related to the
third coagulation phase in plasma, and the prolongation of TT indicates inhibition of throm-
bin-mediated fibrin formation (Dang et al. 2015; Zhang et al. 2016). In this study, the results
implied that compounds 1 and 2 exhibited potential anticoagulant effects by acting on
different coagulation pathways.
3
. Experimental
3
.1. General experimental procedure
NMR spectra were recorded on a Bruker AVANCE-III HD 400 spectrometer. HRESI-MS was
carried out in the positive ion mode with a Bruker microOTOF-Q III spectrometer. Optical
rotations were measured on a polAAr 31 polarimeter. UV spectra were run on a Shimadzu
UV-2700 spectrophotometer. IR spectra were recorded on a PerkinElmer SpectrumTwo spec-
trophotometer in a KBr disc. Column chromatography (CC) was performed on silica gel
(
200–300 mesh, Yantai Institute of Chemical Technology, Yantai, P. R. China), Sephadex LH-20
gel (GE Healthcare Amersham Biosciences, Uppsala, Sweden) and Diaion HP-20 resin
Mitsubishi Chemical Corporation, Tokyo, Japan). TLC analysis was done on pre-coated silica
(
gel GF254 plates (10–40 μm, Yantai Institute of Chemical Technology, Yantai, P. R. China). The
anticoagulant activity was measured on platelet aggregation and blood coagulation factors
analyser (LG-PABER-I, Steellex, Beijing, P. R. China). TT (No. 121167) and PT (No. 105A278) kits

NATURAL PRODUCT RESEARCH
5
were obtained from Sun Biochemical Co. Ltd. (Shanghai, P. R. China). Heparin sodium was
purchased from Macklin Biochemical Co. Ltd. (Shanghai, P. R. China).
3
.2. Material
The leaves of A. argyi were collected in May 2014 from Qichun, Hubei province, People’s
Republic of China. The sample was identified by Prof. Sui-Qing Chen from School of Pharmacy,
Henan University of Traditional Chinese Medicine. A voucher specimen (2014-Y0524) was
deposited at the herbarium of School of Pharmacy, Xinxiang Medical University.
3
.3. Extraction and isolation
The air-dried and milled leaves of A. argyi (12.5 kg) were percolated with 95% EtOH (125 L)
at room temperature. The filtrate was evaporated in vacuum to obtain a residue (2.64 kg),
which was suspended in H O (5 L) and extracted with petroleum ether (6 × 1 L), CH Cl (6 × 1
2
2
2
L) and EtOAc (6 × 1 L), respectively. The EtOAc extract (525 g) was subjected to Diaion HP-20
CC, eluted with EtOH/H O (gradient 80% and 90%) to obtain 80% and 90% EtOH fractions.
2
About 80% EtOH fraction (385 g) was chromatographed over silica gel CC eluting with petro-
leum ether–acetone (gradient 8:1, 4:1, 2:1 and 1:1) and petroleum ether–acetone–MeOH
(
1:1:0.5) to yield 14 fractions 1–14. Fraction 14 (249.3 g) was subjected to Diaion HP-20 col-
umn, eluting with EtOH/H O (gradient 40, 60, 80 and 90%) to obtain subfractions 14.1–14.4.
2
Subfraction 14.1 (40% EtOH fraction, 197.9 g) was separated by CC over silica gel eluted with
CHCl –MeOH (30:1, 20:1, 15:1, 10:1, 5:1, 2:1, 1:1 and 0:1) to yield 13 subfractions, 14.1.1–
3
1
4.1.13. Compound 1 (17.5 mg) was afforded from subfraction 14.1.11 by repeating CC on
silica gel. Subfraction 14.1.8 was repeatedly chromatographed over silica gel and purified
by Sephadex LH-20 CC (CH Cl /MeOH, 1:1) to afford compound 2 (4.6 mg).
2
2
3
.3.1. Eupatilin 7-O-β-d-glucopyranoside (1)
20
Yellowish amorphous powder. [ꢀ]D − 64 (c 0.05, MeOH). UV (MeOH) λ (log ε): 214 (4.99),
max
−1
2
1
5
41 (sh) (4.56), 250 (4.57), 275 (4.62), 338 (4.77). IR (KBr) νmax cm : 3380, 2936, 1662, 1614,
+
465, 1367, 1268, 1072, 814, 686. HRESI-MS: m/z 529.1313 [M + Na] (Calcd for C H O Na,
2
4
26 12
1
29.1322). H NMR (DMSO-d , 400 MHz): δ 7.07 (1H, s, H-3), 7.08 (1H, s, H-8), 7.59 (1H, d,
6
H
J = 1.4 Hz, H-2′), 7.15 (H, d, J = 8.6 Hz, H-5′), 7.71 (H, dd, J = 8.6, 1.4 Hz, H-6′), 5.13 (1H, over-
lapped, H-1″), 3.35 (1H, overlapped, H-2″), 3.30 (1H, overlapped, H-3″), 3.19 (1H, m, H-4″),
3
.47 (1H, overlapped, H-5″), 3.74 (1H, m, H -6″), 3.50 (1H, overlapped, H -6″), 3.78 (3H, s,
a
b
OCH -6), 3.88 (3H, s, OCH -3′), 3.86 (3H, s, OCH -4′), 12.92 (1H, s, OH-5), 5.48 (1H, d, J = 4.9 Hz,
3
3
3
OH-2″), 5.19 (1H, d, J = 4.3 Hz, OH-3″), 5.13 (1H, overlapped, OH-4″), 4.68 (1H, t, J = 5.2 Hz,
1
3
OH-6″). C NMR (DMSO-d , 100 MHz): δ 163.9 (C-2), 103.7 (C-3), 182.5 (C-4), 152.3 (C-5),
6
C
132.6 (C-6), 156.6 (C-7), 94.7 (C-8), 152.2 (C-9), 105.8 (C-10), 122.8 (C-1′), 109.5 (C-2′), 149.1
(C-3′), 152.5 (C-4′), 117.7 (C-5′), 120.2 (C-6′), 100.4 (C-1″), 73.2 (C-2″), 76.8 (C-3″), 69.7 (C-4″),
7
7.5 (C-5″), 60.7 (C-6″), 60.3 (OCH -6), 55.9 (OCH -3′), 55.8 (OCH -4′).
3 3 3
3
.3.2. 5,6,2′,4′-tetrahydroxy-7,5′-dimethoxyflavone (2)
Yellow amorphous powder. UV (MeOH) λ (log ε): 211 (4.81), 242 (sh) (4.31), 262 (sh) (4.40),
max
−1
283(4.47), 367(4.61) nm. IR (KBr) νmax cm : 3375, 2925, 1663, 1603, 1428, 1360, 1267, 1203,
+
1
1118, 860, 797, 565. HRESI-MS: m/z 369.0584 [M + Na] (Calcd for C H O Na, 369.0586). H
1
7
14
8

6
J.ꢀL. LV ET AL.
NMR (DMSO-d , 400 MHz): δ 7.08 (1H, s, H-3), 6.94 (1H, s, H-8), 6.57 (1H, s, H-3′), 7.45 (H, s,
6
H
H-6′), 3.93 (3H, s, OCH -7), 3.82 (3H, s, OCH -5′), 12.78 (1H, s, OH-5), 8.65 (1H, br s, OH-6), 10.42
3
3
1
3
(
1H, br s, OH-2′), 10.03 (1H, br s, OH-4′). C NMR (DMSO-d , 100 MHz): δ 161.7 (C-2), 106.7
6 C
(C-3), 182.2 (C-4), 146.1 (C-5), 129.7 (C-6), 154.2 (C-7), 91.2 (C-8), 149.6 (C-9), 104.8 (C-10), 107.1
(
C-1′), 152.9 (C-2′), 104.4 (C-3′), 151.7 (C-4′), 141.6 (C-5′), 111.9 (C-6′), 56.3 (OCH -7), 56.7
3
(
OCH -5′).
3
3
.4. Acid hydrolysis of compound 1
A solution of 1 (1.9 mg) in MeOH (3.0 mL) was treated with 2.0 M HCl (5.0 mL) and refluxed
for 4 h at 100°C. After removal of the solution under vacuum, 5 mL of distilled water were
added, and then partitioned with 5 mL of ethyl acetate (EtOAc) thrice. The aqueous layer
was neutralised with NaHCO and condensed to dryness. The residue was dissolved in 1 mL
3
of distilled water and was analysed by co-TLC using the solvent system, CHCl –MeOH–H O
3
2
(
2:1:0.1). The TLC plate was sprayed with 10% H SO for detection. Comparison of the reten-
2 4
tion factor value (R ) in the TLC and optical rotation of the sugar in aqueous layer with those
f
of the authentic sugar confirmed that the sugar in 1 was d-glucose (Xiao et al. 2016).
3
.5. Anticoagulation activities assay
Male New Zealand white rabbits (Animal Experimental Center of Zhengzhou University,
Zhengzhou, China) weighing 2.0 ± 0.2 kg were used. The animal experimental procedures
were performed in accordance with the permission of the animal ethical committee of
Xinxiang Medical University. The fresh blood samples were acquired from the common
carotid artery and mixed with 3.8% sodium citrate (blood/citrate: 9/1, v/v), then the mixture
was centrifuged at 3000 rpm for 10 min to obtain plasma (Zhang et al. 2016).
The samples were dissolved in DMSO, and DMSO was used as a control group. Heparin
sodium was used as positive control. The LG-PABER-I coagulation analysis instrument was
used to estimate TT and PT. TT was measured by incubating 50 μL of plasma with 10 μL of
compound for 3 min at 37°C. Then 50 μL of thrombin was added, and the clotting time was
recorded. PT was measured by incubating 50 μL of plasma with 10 μL of compound for 5 min
at 37°C. Then 100 μL of warmed thromboplastin agent was added, and the clotting time was
recorded. All the results were expressed as mean ± standard deviation, three times in four
channels. Statistical significance was determined with t-test. The P-values of less than 0.05
(
p < 0.05) were considered significant.
4
. Conclusion
Our present work on the leaves of A. argyi yielded two new flavonoids (1 and 2). Their struc-
tures were elucidated on the basis of extensive spectroscopic analysis. Compound 1 signif-
icantly extended TT. Compound 2 had obvious effect in increasing PT. The both new
compounds exhibited potential anticoagulation activities through different pathways.
Supplementary material
HRMS and NMR spectra of compounds 1 and 2 are available online.

NATURAL PRODUCT RESEARCH
7
Disclosure statement
No potential conflict of interest was reported by the authors.
Funding
This work was supported by the National Natural Science Foundation of China [grant number 31400305],
grant number 81202916]; the Science and Technology Project of the Education Department of Henan
[
Province [grant number 14A360012]; the Project for Youth Backbone Teachers in the University of
Henan Province [grant number 2015GGJS-130]; the Science and Technology Project of Henan Province
[grant number 152102310113].
References
Allison BJ, Allenby MC, Bryant SS, Min JE, Hieromnimon M, Joyner PM. 2017. Antibacterial activity of
fractions from three Chumash medicinal plant extracts and in vitro inhibition of the enzyme enoyl
reductase by the flavonoid jaceosidin. Nat Prod Res. 31:707–712.
Dang X, Miao JJ, Chen AQ, Li P, Chen L, Liang JR, Xie RM, Zhao Y. 2015. The antithrombotic effect of
RSNK in blood-stasis model rats. J Ethnopharmacol. 173:266–272.
Ge YB, Wang ZG, Xiong Y, Huang XJ, Mei ZN, Hong ZG. 2016. Anti-inflammatory and blood stasis
activities of essential oil extracted from Artemisia argyi leaf in animals. J Nat Med. 70:531–538.
Ji S, Lu GR, Meng DL, Li N, Li X. 2010. Chemical constituents from the Folium Artemisiae Argyi (II). J
Shenyang Pharm Univ. 27:548–550.
Lee SH, Kim HK, Seo JM, Kang HM, Kim JH, Son KH, Lee H, Kwon BM. 2002. Arteminolides B, C, and
D, new inhibitors of farnesyl protein transferase from Artemisia argyi. J Org Chem. 67:7670–7675.
Lee HG, Yu KA, Oh WK, Baeg TW, Oh HC, Ahn JS, Jang WC, Kim JW, Lim JS, Choe YK, Yoon DY. 2005.
Inhibitory effect of jaceosidin isolated from Artemisia argyi on the function of E6 and E7 oncoproteins
of HPV 16. J Ethnopharmacol. 98:339–343.
Liu XH, Zhou A, Liu BS, Chen HJ, Shen DK. 2006. Bacteriostatic effect of volatile oil from Artemisia Argyi
in vitro and in vivo. Chin J Inform TCM. 13:25–26.
Lv JL, Duan JA, Shen B, Yin YY. 2013. Caffeic acid esters from Artemisia argyi and their antioxidant
activities. Chem Nat Compd. 49:8–11.
Nakasugi T, Nakashima M, Komai K. 2000. Antimutagens in gaiyou (Artemisia argyi Levl. et Vant.). J
Agric Food Chem. 48:3256–3266.
Ngankeu Pagning AL, Tamokou JDD, Lateef M, Tapondjou LA, Kuiate JR, Ngnokam D. 2016. New
triterpene and new flavone glucoside from Rhynchospora corymbosa (Cyperaceae) with their
antimicrobial, tyrosinase and butyrylcholinesterase inhibitory activities. Phytochem Lett. 16:121–128.
Ornano L, Venditti A, Donno Y, Sanna C, Ballero M, Bianco A. 2016. Phytochemical analysis of non-
volatile fraction of Artemisia caerulescens subsp. densiflora (Viv.) (Asteraceae), an endemic species
of La Maddalena Archipelago (Sardinia – Italy). Nat Prod Res. 30:920–925.
Peron G, Baldan V, Sut S, Faffian M, Roccabruna L, Zanini D, Manzini P, Maggi F, Dall’Acqua S. 2017.
PhytochemicalinvestigationsonArtemisiaalbaTurragrowingintheNorth-EastofItaly.NatProdRes.doi:
10.1080/14786419.2016.1263845.
Wang S, Li J, Sun J, Zeng KW, Cui JR, Jiang Y, Tu PF. 2013. NO inhibitory guaianolide-derived terpenoids
from Artemisia argyi. Fitoterapia. 85:169–175.
Wang S, Sun J, Zeng KW, Chen XG, Zhou WQ, Zhang C, Jin HW, Jiang Y, Tu PF. 2014. Sesquiterpenes
from Artemisia argyi: absolute configurations and biological activities. Eur J Org Chem. 5:973–983.
Wu MY, Wen DD, Gao N, Xiao C, Yang L, Xu L, Lian W, Peng WL, Jiang JM, Zhao JH. 2015. Anticoagulant
and antithrombotic evaluation of native fucosylated chondroitin sulfates and their derivatives as
selective inhibitors of intrinsic factor Xase. Eur J Med Chem. 92:257–269.
Xiao YY, Xie HH, Zhao L, Gou P. 2016. Acyl flavone and lignan glucosides from Leontopodium
leontopodioides. Phytochem Lett. 17:247–250.

8
J.ꢀL. LV ET AL.
Xin N, Li YJ, Li Y, Dai RJ, Meng WW, Chen Y, Schlappi M, Deng YL. 2011. Dragon’s blood extract has
antithrombotic properties, affecting platelet aggregation functions and anticoagulation activities.
J Ethnopharmacol. 135:510–514.
Xu XQ, Liu WJ, Li WZ, Liu SW. 2016. Anticoagulant activity of crude extract of Holotrichia diomphalia
larvae. J Ethnopharmacol. 177:28–34.
Zeng KW, Wang S, Dong X, Jiang Y, Tu PF. 2014. Sesquiterpene dimer (DSF-52) from Artemisia argyi
inhibits microglia-mediated neuroinflammation via suppression of NF-κB, JNK/p38 MAPKs and Jak2/
Stat3 signaling pathways. Phytomedicine. 21:198–306.
Zhang LB, Lv JL, Chen HL, Yan XQ, Duan JA. 2013. Chemical constituents from Artemisia argyi and their
chemotaxonomic significance. Biochem Syst Ecol. 50:455–458.
Zhang LB, Lv JL, Liu JW. 2016. Phthalide derivatives with anticoagulation activities from Angelica sinensis.
J Nat Prod. 79:1857–1861.
Zhong YR, Cui SL. 1992. Effective chemical constituents of Artemisia argyi Levl. et Vant for inhibition of
platelet aggregation. Chin J Chin Mater Med. 17:353–354.