Butenolides, triterpenoids and phenylethanoid glycosides from Plantago depressa

Article abstract of DOI:10.1016/j.phytol.2019.01.002

Four new compounds, including two butenolides (1 and 2), an arborinane-type triterpenoid (3) and a phenylethanoid glycoside (4), along with the known triterpenoids (5–17) and phenylethanoid glycosides (18–29), were isolated from whole plants of Plantago depressa. The structures of the isolated compounds were elucidated by comprehensive spectroscopic analyses and were evaluated for their anti-inflammatory or antiradical activities. Our results underlined that, among compounds 1–17, only compound 16 inhibited lipopolysaccharide-induced NO production with an IC₅₀ equal to 5.33 ± 0.51 μM. All phenylethanoid glycosides (4, 18–29) were evaluated for their antiradical activity with a DPPH radical scavenging bioassay and showed high to moderate effect (IC₅₀ from 2.70 to 51.51 μM).

Full text of DOI:10.1016/j.phytol.2019.01.002

Phytochemistry Letters 30 (2019) 21–25
Contents lists available at ScienceDirect
Phytochemistry Letters
journal homepage: www.elsevier.com/locate/phytol
Butenolides, triterpenoids and phenylethanoid glycosides from Plantago
depressa
a,b,1
b
b,1
a,b
b
b
b
Xiu-Qing Song
, Jie Bao , Jia Sun , Zhi-Qiang Cheng , Yuying Zhang , Jin-Hai Yu ,
b,⁎
Cheng-Shi Jiang , Hua Zhang
a
b
School of Chemistry and Chemical Engineering, University of Jinan, 336 West Road of Nan Xinzhuang, Jinan 250022, China
School of Biological Science and Technology, University of Jinan, 336 West Road of Nan Xinzhuang, Jinan 250022, China
A R T I C L E I N F O
A B S T R A C T
Keywords:
Four new compounds, including two butenolides (1 and 2), an arborinane-type triterpenoid (3) and a pheny-
lethanoid glycoside (4), along with the known triterpenoids (5–17) and phenylethanoid glycosides (18–29),
were isolated from whole plants of Plantago depressa. The structures of the isolated compounds were elucidated
by comprehensive spectroscopic analyses and were evaluated for their anti-inflammatory or antiradical activ-
ities. Our results underlined that, among compounds 1–17, only compound 16 inhibited lipopolysaccharide-
induced NO production with an IC50 equal to 5.33 ± 0.51 μM. All phenylethanoid glycosides (4, 18–29) were
evaluated for their antiradical activity with a DPPH radical scavenging bioassay and showed high to moderate
effect (IC50 from 2.70 to 51.51 μM).
Plantago depressa
Butenolide
Triterpenoid
Phenylethanoid glycoside
Anti-inflammation
Antiradical
1
. Introduction
the sugar moiety in 4 was determined by chiral HPLC comparison of its
hydrolysis derivatives with prepared standard samples. Some com-
Plantago depressa Willd. is an herbal plant commonly found in Asia;
pounds showed anti-inflammatory and antiradical properties. Herein,
this paper describes the isolation, structure characterization and bio-
logical evaluation of these natural products.
both its seeds and the plant as a whole are used in China for traditional
therapies, as they have a variety of medicinal properties, such as
clearing internal heat and eliminating phlegm (Flora of China Editorial
Committee of Chinese Academy of Sciences, 2002; National
Pharmacopoeia Committee, 2015). P. depressa is rich in phenylethanoid
glucosides (Yan et al., 2009; Feng et al., 2017), iridoid glucosides
2. Results and discussion
Compound 1 was assigned the molecular formula of C18
H
30
O by
5
+
(
Zhang et al., 1996; Sun et al., 2010) and polysaccharides (Olennikov
(+)-HR-ESIMS analysis at m/z 349.1983 ([M + Na] , calcd 349.1985),
1
et al., 2011), according to published reports. Our preliminary chemical
investigation has revealed the presence of lipid constituents in the
ethanolic extract of this plant, and a subsequent fractionation of the
ethyl acetate sub-extract returned 19 fatty acid derivatives (Song et al.,
indicating four degrees of unsaturation. The H NMR data (Table 1)
revealed the presence of a methoxyl [δ
H
H
3.67 (s)], two methyl [δ 1.82
H
(s), 1.94 (s)], and ten methylene [δ
1.16 (1H, m), 1.32 (1H, m),
1.22–1.35 (12H, m), 1.61 (2H, m), 1.75 (1H, m), 1.97 (1H, m), 2.30
1
3
2
018). Apart from the abovementioned lipid molecules, an intensive
(2H, t, J = 7.5 Hz)] groups. The C NMR data (Table 1) showed signals
3
study on the remaining fractions led to the separation and identification
of two new butenolides (1 and 2), a new arborinane triterpenoid (3)
and 13 known triterpenoid analogues (5–17) (Fig. 1). Additionally, the
polar constituents in the n-BuOH partition were further investigated,
and 13 phenylethanoid glycosides, including one new glycoside (4) and
of two carbonyl (δ
C
174.6, 172.0), two olefinic (δ 157.7, 125.5), a sp
C
quaternary (δ
C
106.9), a methoxyl (δ
C
51.6), two methyl (δ
C
10.9, 8.6)
and ten methylene (δ
C
36.2–23.0) carbons. These observations ac-
counted for three degrees of unsaturation and required a monocyclic
scaffold for 1. The aforementioned spectral features were similar to
those of 13-(2-hydroxy-3,4-dimethyl-5-oxo-2,5-dihydrofuran-2-yl) tri-
decanoic acid methyl ester isolated from the brittle star Ophiomastix
mixta. (Lee et al., 2007), but with two less methylene groups, which was
1
2 known structurally related glycosides (18–29) (Fig. 1), were ob-
tained. The structures of these compounds were elucidated by com-
prehensive spectroscopic analyses, and the absolute configuration of
⁎
Corresponding author.
E-mail address: bio_zhangh@ujn.edu.cn (H. Zhang).
1
These authors contributed equally.
https://doi.org/10.1016/j.phytol.2019.01.002
Received 3 August 2018; Received in revised form 14 December 2018; Accepted 2 January 2019
1874-3900/ © 2019 Published by Elsevier Ltd on behalf of Phytochemical Society of Europe.

X.-Q. Song et al.
Phytochemistry Letters 30 (2019) 21–25
Fig. 2. 2D NMR correlations for 1.
to C-2, C-3 and C-4 (δ
C
106.9), and H
2
-14 and OCH
3
to C-15 (δ 174.6).
C
Thus, the structure of 1 was characterized as 11-(2-hydroxy-3,4-di-
methyl-5-oxo-2,5-dihydrofuran-2-yl) hendecanoic acid methyl ester,
with only one chiral centre. Further spectroscopic analyses revealed a
near zero [α] value and a smooth ECD curve for 1, indicating that it
D
might be a racemic mixture. However, neither normal phase nor re-
versed phase chiral HPLC analysis provided decent resolution for fur-
ther separation. The absolute configuration of 1 thus remained unas-
signed.
Compound 2 was assigned the molecular formula of C19
H
32
O by
5
+
(
+)-HR-ESIMS analysis at m/z 363.2143 ([M + Na] , calcd 363.2142),
1
13
having 14 more mass units than 1. The H and C NMR data of 2
Table 1) were highly similar to those of 1, except that the methoxyl
group [δ 3.67 (s); δ 51.6] in 1 was replaced by an ethoxyl moiety [δ
.11 (2H, q, J = 7.2 Hz), 1.24 (3H, t, J = 7.2 Hz); δ 60.4, 14.4.] in 2.
(
H
C
H
4
C
This assignment was further supported by careful examination of the 2D
NMR data. Therefore, the structure of 2 was identified as 11-(2-hy-
droxy-3,4-dimethyl-5-oxo-2,5-dihydrofuran-2-yl) hendecanoic acid
ethyl ester. As in the case of compound 1, the absolute stereochemistry
of 2 was not able to be established, and it could be an artefact from
interesterification reaction from 1 due to extraction in EtOH.
Compound 3 was assigned the molecular formula of C32
H
50
O by
5
+
(
+)-HR-ESIMS analysis at m/z 537.3554 ([M + Na] , calcd 537.3550).
−
1
The IR spectrum exhibited absorption bands for hydroxyl (3423 cm ),
Fig. 1. Chemical structures of 1–4 from Plantago depressa.
−1
−1
carbonyl (1728 cm ), and olefinic (1641 cm ) groups. Detailed
analysis of the NMR data (Table 2) for 3 revealed high similarity to
those of rubiarbonone B (Xu, 1995), with the only difference being
Table 1
1
H (600 MHz) and 13C (150 MHz) NMR data for 1 and 2 in CDCl
3
.
attributed to the resonances of an additional acetyl group [δ 2.05 (3H,
H
No.
1
2
s), δ
downfield shifted H-7 signal (δ
H-7 to a carbonyl at δ
C
170.4] in 3. Compared with rubiarbonone B, the significantly
H
4.99) of 3 and the HMBC correlation of
δ
H
δ
C
δ
H
δ
C
C
170.1 (Fig. 3) corroborated the location of the
1
2
3
4
5
172.0
157.7
125.5
106.9
36.2
172.2
157.9
125.4
107.1
36.1
acetoxyl group at C-7. Further inspection of proton-proton couplings
and NOESY data (Fig. 3) defined the relative configuration of 3 as
shown, being the same as that of rubiarbonone B. Thus, compound 3
was characterized as 7-O-acetylrubiarbonone B.
1.75, m
1.75, m
1.97, m
1.14, m,
1.30, m
Compound 4 was assigned the molecular formula of C29
H
36
O14 by
1
.97, m
−
6
1.16, m
23.0
23.0
(−)-HR-ESIMS analysis at m/z607.2034 ([M−H] , calcd 607.2032).
1
.32, m
−1
The IR spectrum indicated the presence of hydroxyl (3392 cm ),
a
c
7
-10
1.22–1.35 , m
29.40
1.21–1.31 , m
29.4
29.45
29.45
29.47
29.3
29.2
25.1
34.5
174.2
8.6
−1
-1
conjugated ester (1699 cm ) and aromatic (1605, 1524 cm ) func-
tionalities. The NMR data (Table 2) for 4 revealed high similarity to
those of its co-metabolite plantamajoside (18) (Ravn et al., 1990), and
the only difference was attributed to the signals for the 3,4-dihydrox-
yphenethyl moiety in the latter being replaced by those for a phenethyl
b
b
b
d
d
d
2
2
2
9.45
9.45
9.46
a
a
c
c
1
1
1
1
1
1
1
1
2
3
4
5
6
7
1.22–1.35 , m
29.3
29.2
25.1
34.3
174.6
8.6
1.21–1.31 , m
1.22–1.35 , m
1.21–1.31 , m
1.61, m
1.60, m
moiety [δ 7.26–7.28 (4 H), 7.18 (1H, m)] in 4. This was also supported
H
2.30, t (7.5)
2.28, t (7.5)
by their MS differences (32 mass units) and further confirmed by de-
1
1
1.82, s
1.94, s
3.67, s
1.81, s
1.93, s
tailed interpretation of 2D H- H COSY and HMBC correlations (Fig. 4).
10.9
51.6
10.9
The large coupling constants (JH-3, 4 = 9.3 Hz and JH-3', 4' = 9.3 Hz) in
OMe
OEt
1
H NMR indicated that 3/3'-OH and 4/4'−OH, respectively, were in
4.11, q (7.2)
60.4
14.4
different orientations (Reddy et al., 1998), implying the sugar in 4 was
glucose rather than galactose. This was also proven by the chemical
shift value (105.8) for C-1' in 4 (nearly 100 for galactose and nearly 105
for glucose) (Wang et al., 2009). The relative configurations of the
sugar anomeric carbons were both assigned to be β from the coupling
constants of 7.9 and 7.7 Hz. Both sugars were determined to be D-
glucose by comparing the HPLC chromatograms of the acetylated pro-
ducts from acidic hydrolysis of 4 with those of the acetylated products
from standard D- and L-glucoses (Fig. S30, Supporting information).
1
.24, t (7.2)
a,cOverlapping signals; b,dInterchangeable assignments.
also supported by their MS differences (28 mass units). Detailed ana-
lyses of 2D NMR data (Fig. 2) further confirmed that 1 was an analogue
of the abovementioned known compound, only with a two-carbon
shorter side chain. The key HMBC correlations included those from H -
3
1
6 (δ
H
1.82) to C-1 (δ
C
172.0), C-2 (δ
C
157.7) and C-3 (δ
C
125.5), H -17
3
22

X.-Q. Song et al.
Phytochemistry Letters 30 (2019) 21–25
Table 2
1
13
H (600 MHz) and C (150 MHz) NMR data for 3 and 4.
a
b
No.
3
No.
4
δ
H
δ
C
δ
H
δ
C
1
2
1.75, ddd (14.2, 13.2, 4.7) 36.9
1
2
3
4
5
6
4.44, d (7.9)
3.49, m
104.0
76.0
84.2
70.9
75.1
2.06, m
2.40, ddd (15.4, 4.7, 3.0)
.74, ddd (15.4, 14.2, 5.8)
34.9
3.96, dd (9.3, 9.3)
4.93, dd (9.4, 9.4)
3.49, m
2
3
4
5
215.9
47.4
48.6
3.55, dd (11.5, 5.6) 62.4
3.65, brd (11.5)
1.39, m
1
′
4.54, d (7.7)
105.8
76.0
77.7
71.2
78.0
62.3
6
1.98, ddd (12.5, 5.8, 2.5)
29.0
2′
3′
4′
5′
3.14, dd (9.3, 7.7)
c
1
.65, m
3.32 , dd (9.3, 9.3)
7
8
9
4.99, ddd (10.5, 10.5, 5.8) 73.6
3.22, dd (9.3, 9.3)
3.16, m
2.48, m
45.1
144.2 6′
39.0
3.49, m
1
1
1
0
1
2
3.68, dd (11.8, 2.4)
5.43, ddd (6.4, 2.1, 2.1)
2.08, m
119.1 1′′
140.0
129.4
130.1
127.2
130.1
129.4
37.2
d
37.0
2′′
3′′
4′′
5′′
6′′
7′′
8′′
7.26–7.28
d
1
.85, m
7.26–7.28
1
1
1
3
4
5
37.6
39.8
32.0
7.18, m
d
7.26–7.28
d
1.55, m
.28, m
1.39, m
.68, m
7.26–7.28
1
2.96, m
3.79, m
4.12, m
1
6
32.7
71.9
1
1
1
1
2
7
8
9
0
48.8
59.6
71.3
42.8
1′′′
2′′′
3′′′
4′′′
5′′′
6′′′
7′′′
8′′′
9′′′
127.7
115.2
149.7
146.9
116.5
123.1
147.4
115.2
168.5
1.83, d (9.8)
4.46, m
7.06, d (2.1)
Fig. 3. 2D NMR correlations for 3.
1.67, m
2
.12, ddd (14.2, 10.6, 9.4)
6.78, d (8.2)
2
2
2
2
2
2
2
2
1
2
3
4
5
6
7
8
1.40, m
56.9
30.8
25.2
22.1
21.2
17.1
16.4
63.9
6.98, dd (8.2, 2.1)
7.58, d (15.9)
6.32, d (15.9)
1.64, m
1.06, s
1.04, s
1.30, s
0.96, s
1.07, s
3.78, dd (11.9, 5.0)
3
.88, dd (11.9, 5.0)
2
3
9
0
0.95, d (6.4)
0.88, d (6.5)
22.9
23.3
170.4
22.1
OAc
Fig. 4. 2D NMR correlations for 4.
2
.05, s
2
8-OH 1.08, t (5.0)
a
b
c
Table 3
Antiradical assay results for 4 and 18–19.
In CDCl
3
.
In CD
3
OD.
Overlapped by solvent peak.
No.
IC50 (μM)
No.
IC50 (μM)
d
Overlapping signals.
4
37.67 ± 1.80
51.51 ± 4.26
15.06 ± 3.67
15.53 ± 2.10
2.70 ± 0.48
4.76 ± 1.53
14.57 ± 2.38
24
5.90 ± 2.91
18.57 ± 1.24
5.43 ± 2.67
12.22 ± 6.34
47.00 ± 7.61
8.56 ± 1.81
7.80 ± 0.51
1
8
25
The structure of 4 was thereby elucidated.
19
20
26
The known compounds were identified, on the basis of detailed
spectroscopic interpretation, to be ursolic acid lactone (5) (Al Musayeib
et al., 2013), rubiarbonol A (6) (Itokawa et al., 2008), rubiarbonol B (7)
27
2
2
2
1
2
3
28
29
ascorbic acid
(
(
(
Itokawa et al., 2008), rubiarbonol L (8) (Fan et al., 2011), ursolic acid
9) (Seebacher et al., 2003), pomolic acid (10) (Lee et al., 2005), uvaol
11) (Zhao et al., 2014), corosolic acid (12) (Saimaru et al., 2007),
glycosides (4 and 18–29), and all tested compounds showed high to
moderate activity (Table 3). A preliminary structure-activity-relation-
ship analysis revealed that those possessing more phenolic hydroxyls
displayed better activity (Lv et al., 2018).
spinosic acid (13) (Wang et al., 2011), oleanolic acid (14) (Seebacher
et al., 2003), maslinic acid (15) (Saimaru et al., 2007), olean-12-en-2,3-
diol (16) (Braca et al., 2001), erythrodiol (17) (Saimaru et al., 2007),
plantamajoside (18) (Ravn et al., 1990), plontoinoside D (19) (Miyase
et al., 1991), jionoside C (20) (Sasaki et al., 1989), acteoside (21) (Wu
et al., 2003), isoacteoside (22) (Nishimura et al., 1991), jionoside D
3. Materials and methods
(
23) (Sasaki et al., 1989), stachysoside A (24) (Nishimura et al., 1991),
plonrainoside
C
(25) (Miyase et al., 1991), cis-acteoside (26)
3.1. General experimental procedures
(
(
Nishimura et al., 1991), salsaside B (27) (Lei et al., 2010), martynoside
28) (Sasaki et al., 1989) and suspensaside B (29) (Ming et al., 1999).
IR spectra were recorded on an FT-IR VERTEX 70 instrument
(Bruker BioSpin AG, Fremont, USA). Optical rotations were measured
Anti-inflammatory assays in mouse macrophage RAW264.7 cells
were conducted for compounds 1–17. Only compound 16 exhibited NO
production inhibition with an IC50 value of 5.33 ± 0.51 μM. The DPPH
radical scavenging ability was evaluated for all phenylethanoid
on
a
Rudolph VI polarimeter (Rudolph Research Analytical,
Hackettstown, NJ, USA) with a 10 cm length cell. NMR experiments
were recorded on a Bruker Avance DRX600 spectrometer (Bruker
23

X.-Q. Song et al.
Phytochemistry Letters 30 (2019) 21–25
BioSpin AG, Fallanden, Switzerland) and referenced to residual solvent
Compound 1: colourless oil; [α]25 D –0.8 (c 0.1, CH Cl); UV
3
peaks (CD
3
OD: δ
H
3.31, δ
C
49.00; CDCl
: δ
3 H
7.26, δ
C
77.16). ESIMS
(MeOH) λmax (log ε) 272 (3.75); IR (KBr) νmax 3435, 2931, 2862, 1743,
−1 1 13
analyses were carried out on an Agilent 1260–6460 Triple Quad LC–MS
instrument (Agilent Technologies Inc., Waldbronn, Germany). HR-
ESIMS spectra were obtained on an Agilent 6545 Q-TOF mass spec-
trometer (Agilent Technologies Inc., Waldbronn, Germany). UV spectra
were obtained on a Shimadzu UV-2600 spectrophotometer (Shimadzu,
Kyoto, Japan) with a 1 cm pathway cell. Normal HPLC separation was
performed using an Agilent 1260 series LC instrument (Agilent
Technologies Inc., Waldbronn, Germany) coupled with an Agilent SB-
C18 column (9.4 × 250 mm, Agilent Technologies Inc., Santa Clara,
CA, USA). Chiral MZ(2) RH 5 u column (4.6 × 250 mm, Phenomenex,
Washington, D.C., USA) and CHIRALPAK AD-H column (4.6 × 250 mm,
Daicel Corporation, Tokyo, Japan) were used for chiral HPLC analysis.
Column chromatography (CC) was performed on D101-macroporous
absorption resin (Sinopharm Chemical Reagent Co. Ltd., Shanghai,
China), MCI gel (CHP20 P, Mitsubishi Chemical Corporation, Tokyo,
Japan), reversed phase (RP) C18 silica gel (Merck KGaA, Darmstadt,
Germany), Sephadex LH-20 (GE Healthcare Bio-Sciences AB, Uppsala,
Sweden) and silica gel (300–400 mesh; Qingdao Marine Chemical Co.
Ltd., Qingdao, China). All solvents used for CC were of analytical grade
1385, 1258, 1051 cm ; for H and C NMR data, see Table 1;
+
(+)-ESIMS m/z 349.1 [M + Na] ; (+)-HR-ESIMS m/z 349.1983 [M +
+
Na] (calcd for C18
H
30
O Na, 349.1985).
5
Compound 2: colourless oil; [α]25 D –0.5 (c 0.05, CH Cl); UV
3
(MeOH) λmax (log ε) 273 (3.95); IR (KBr) νmax 3422, 2935, 2862, 1734,
−1
1
13
1439, 1383, 1180, 1120, 958 cm ; for H C NMR data, see Table 1;
+
(+)-ESIMS m/z 363.2 [M + Na] ; (+)-HR-ESIMS m/z 363.2143 [M +
+
Na] (calcd for C19
H
32
5
O Na, 363.2142).
Compound 3: white amorphous powder; [α]25 D –5.0 (c 0.08,
CH Cl); IR (KBr) νmax 3435, 2926, 2858, 1735, 1709, 1625, 1385,
−1 1 13
3
1250 cm ; for H and C NMR data, see Table 2; (+)-HR-ESIMS m/z
+
537.3554 [M + Na] (calcd for C32
H
50
O Na, 537.3550).
5
Compound 4: white amorphous powder; [α]25 D –37.1 (c 0.2,
MeOH); UV (MeOH) λmax (log ε) 212 (4.20), 250 (3.91), 329 (4.31); IR
(KBr) νmax 3389, 2930, 1703, 1630, 1605, 1525, 1445, 1281, 1161,
−
1
1
13
1704, 1034, 814 cm ; for H and C NMR data, see Table 2;
−
−
(–)-ESIMS m/z607.1 [M−H] ; (–)-HR-ESIMS m/z607.2034 [M−H]
(calcd for C29
35
H O14, 607.2032).
(
Tianjin Fuyu Fine Chemical Co. Ltd., Tianjin, China) and solvents used
3
.4. Acid hydrolysis of 4 and chiral HPLC analyses
for HPLC were of HPLC grade (Oceanpak Alexative Chemical Ltd.,
Goteborg, Sweden). Pre-coated silica gel GF254 plates (Qingdao Marine
Chemical Co. Ltd., Qingdao, China) were used for thin-layer chroma-
tography (TLC) monitoring.
A solution of 4 (1.5 mg) in 1.0 mol/L HCl (3.0 mL) was stirred at
8
0 °C for 3 h. After removal of excess HCl under reduced pressure, the
residual aqueous mixture was filtered to eliminate aglycones. The re-
maining solution was evaporated in vacuum to afford the mono-
saccharides which were acetylated with acetic anhydride in pyridine at
room temperature for 10 h. Standard D- and L-glucoses were also
acetylated using the same method. Chiral HPLC analyses were carried
out using a Chiral MZ(2)-RH (4.6 × 250 mm) column (Fig. S30 in
Supporting information).
3.2. Plant material
Whole plants of P. depressa were collected in June 2016 at Mount
Kunyu, Shandong Province, and were authenticated by Prof. Jie Zhou
from the University of Jinan. A voucher specimen was deposited at the
School of Biological Science and Technology, University of Jinan
(
Accession number: npmc-007).
3
.5. Anti-inflammatory assay
3
.3. Extraction and isolation
The anti-inflammatory assay was performed as we reported earlier
(
Song et al., 2018), and the initial screening concentration was set as
The air-dried powder of the whole plants of P. depressa (15 kg) was
2
0 μM. Only compounds with > 50% NO inhibition and > 90% cell
viability were further tested for IC50. Then, the concentration ranges of
.63, 1.25, 2.5, 5.0, 10.0 and 20.0 μM were applied for the IC50 mea-
surement of 16. Dexamethasone was used as a positive control
IC50 = 2.30 ± 0.43 μM).
extracted with 95% EtOH at room temperature three times to afford a
crude extract (0.9 kg). The extract was then suspended in 2.0 L water
and partitioned with EtOAc (2.0 L × 3) and n-BuOH (2.0 L × 3). The
EtOAc partition (300 g) was subjected to CC over D101-macroporous
0
(
absorption resin, eluted with EtOH-H O (30%, 50%, 80% and 95%), to
2
afford four fractions (A, B, C and D). Fraction C (80%, 87 g) was sub-
jected to passage over an MCI gel CC, eluted with MeOH-H O (50% to
00%), to yield five subfractions (C1–C5). Compounds 1 (1.0 mg) and 2
0.6 mg) were obtained from subfraction C2 by using silica gel CC and
3
.6. Antiradical assay
2
1
The antiradical assay was evaluated by a DPPH radical scavenging
(
method as we reported previously (Wang et al., 2018), and the initial
screening concentration was set as 100 μM. Only compounds with >
HPLC; compounds 3 (2.0 mg), 5 (1.5 mg), 6 (1.8 mg), 7 (2.1 mg), 8
(
(
1.1 mg), 9 (1.1 mg), 10 (7.0 mg), 12 (0.5 mg), 13 (4.6 mg), 14
3.4 mg), 15 (0.7 mg) and 16 (5.0 mg) were separated from subfraction
5
0% inhibition were further tested for IC50. Then, the concentration
ranges of 0.78, 1.56, 3.13, 6.25, 12.5, 25.0, 50.0 and 100 μM were
applied for the IC50 measurement for all tested compounds. Ascorbic
acid was used as a positive control (IC50 = 7.80 ± 0.51 μM).
C4 by using silica gel CC, RP-C18 silica gel CC and HPLC; compounds
1
1 (7.5 mg) and 17 (5.8 mg) were obtained from subfraction C5 by
using the same separation methods as subfraction C4 (for details, see
Supporting information).
Conflict of interest
The n-BuOH partition (100 g) was subjected to CC over D101-
macroporous absorption resin, eluted with EtOH-H
2
O (30%, 50% and
The authors declare no conflict of interest.
9
0%), to afford three fractions (E, F and G). Fraction F (30 g) was
subjected to silica gel CC, eluted with CHCl
3
-MeOH (15:1 to 1:1, sa-
turated with H
2
O) to afford three sub-fractions (F1–F3). Compounds 4
Acknowledgements
(
2.5 mg), 23 (7.8 mg) and 27 (1.8 mg) were obtained from subfraction
F1 by using HPLC; compound 25 was separated from subfraction F2 by
using silica gel CC, Sephadex LH-20 CC and HPLC; compounds 18
Financial support from the Natural Science Foundation of Shandong
Province (No. JQ201721), the Young Taishan Scholars Program (No.
tsqn20161037) and Shandong Talents Team Cultivation Plan of
University Preponderant Discipline (No. 10027) are greatly acknowl-
edged. We also thank Prof. Jie Zhou for the identification of the plant
materials and Elsevier Webshop for the language editing service.
(
(
18.0 mg), 19 (23.0 mg), 20 (5.0 mg), 21 (20.0 mg), 22 (5.7 mg), 24
3.5 mg), 26 (4.0 mg), 28 (49.0 mg) and 29 (5.2 mg) were purified from
subfraction F3 by using silica gel CC and HPLC (for details, see
Supporting Information).
24

X.-Q. Song et al.
Phytochemistry Letters 30 (2019) 21–25
Appendix A. Supplementary data
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