Antioxidant and cytotoxic lignans from the roots of Bupleurum chinense

Article abstract of DOI:10.1080/10286020.2016.1234456

In the search for biologically active compounds from the roots of Bupleurum chinense D C., phytochemical investigation of its ethanol extract led to the isolation and identification of a new 8-O-4′ neolignan glucoside, saikolignanoside A (1), along with eight known lignans (2–9). Their structures were determined on the basis of IR, UV, HRESIMS, and NMR spectroscopic analyses. The antioxidant and cytotoxic effects of isolated compounds were evaluated in vitro. The isolated compounds (IC₅₀?>?200?μM) did not display 2, 2-diphenyl-1-picrylhydrazyl (DPPH) radical scavenging activity. Whereas compounds 1–2, 5, 7, and 9 exhibited potent 2, 2′-azinobis(3-ethyl-benzothiazoline-6-sulfonic acid) (ABTS) free radical scavenging properties with IC₅₀ values ranging from 8.34 to 15.24?μM, while compounds 3–4, 6, 8 showed moderate properties. In addition, all compounds were evaluated for cytotoxicities against A549, HepG2, U251, Bcap-37, and MCF-7 cell lines. Compounds 5 and 9 (IC₅₀< 51.62?μM) possessed stronger cytotoxic activities against all the tested tumor cell lines, compared with the positive control 5-Fluorouracil.

Full text of DOI:10.1080/10286020.2016.1234456

Journal of Asian Natural Products Research
ISSN: 1028-6020 (Print) 1477-2213 (Online) Journal homepage: http://www.tandfonline.com/loi/ganp20
Antioxidant and cytotoxic lignans from the roots of
Bupleurum chinense
Dan-Qi Li, Di Wang, Le Zhou, Ling-Zhi Li, Qing-Bo Liu, Ying-Ying Wu, Jing-Yu
Yang, Shao-Jiang Song & Chun-Fu Wu
To cite this article: Dan-Qi Li, Di Wang, Le Zhou, Ling-Zhi Li, Qing-Bo Liu, Ying-Ying Wu,
Jing-Yu Yang, Shao-Jiang Song & Chun-Fu Wu (2016): Antioxidant and cytotoxic lignans
from the roots of Bupleurum chinense, Journal of Asian Natural Products Research, DOI:
10.1080/10286020.2016.1234456
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Date: 27 September 2016, At: 07:41

Journal of asian natural Products research, 2016
http://dx.doi.org/10.1080/10286020.2016.1234456
Antioxidant and cytotoxic lignans from the roots of
Bupleurum chinense
Dan-Qi Lia,b, Di Wanga,b, Le Zhoua,b, Ling-Zhi Lia,b, Qing-Bo Liua,b, Ying-Ying Wua,b
Jing-Yu Yangc, Shao-Jiang Songa,b and Chun-Fu Wuc
,
aschool of traditional chinese Materia Medica, shenyang Pharmaceutical university, shenyang 110016,
china; bKey laboratory of structure-Based drug design and discovery, Ministry of education, shenyang
Pharmaceuticaluniversity, shenyang 110016, china; cdepartmentofPharmacology, shenyang Pharmaceutical
university, shenyang 110016, china
ABSTRACT
ARTICLE HISTORY
received 18 March 2016
accepted 5 september 2016
In the search for biologically active compounds from the roots of
Bupleurum chinense D C., phytochemical investigation of its ethanol
extract led to the isolation and identification of a new 8-O-4′ neolignan
glucoside, saikolignanosideA(1), alongwitheightknownlignans(2–9).
Their structures were determined on the basis of IR, UV, HRESIMS, and
NMR spectroscopic analyses. The antioxidant and cytotoxic effects of
isolated compounds were evaluated in vitro. The isolated compounds
(IC50 > 200 μM) did not display 2, 2-diphenyl-1-picrylhydrazyl (DPPH)
radical scavenging activity. Whereas compounds 1–2, 5, 7, and 9
exhibited potent 2, 2′-azinobis(3-ethyl-benzothiazoline-6-sulfonic
acid) (ABTS) free radical scavenging properties with IC50 values
ranging from 8.34 to 15.24 μM, while compounds 3–4, 6, 8 showed
moderate properties. In addition, all compounds were evaluated
for cytotoxicities against A549, HepG2, U251, Bcap-37, and MCF-7
cell lines. Compounds 5 and 9 (IC50<51.62 μM) possessed stronger
cytotoxic activities against all the tested tumor cell lines, compared
with the positive control 5-Fluorouracil.
KEYWORDS
Bupleurum chinense; lignans;
antioxidant activities;
cytotoxicities; structural
elucidation
1. Introduction
e roots of Bupleurum chinense D C. (Umbelliferae), first recorded in Shen Nong’s Herbal
Classic, has been used for more than 2000 years [1–3]. It also has been widely used as
a key ingredient for many Chinese multi-herb remedies, such as Xiao-chaihu-tang, a
famous hematopoietic remedy in oriental medicine [3]. According to the new Chinese
Pharmacopoeia, the roots of B. chinense have been widely used to treat influenza, fever,
inflammation, malaria, cancer and menstrual disorders [4,5]. Previous phytochemical stud-
ies on B. chinense have led to a series of highly functionalized saikosaponins, flavonoids,
lignans, coumarins, polysaccharides, polyacetylenes, and volatile oil [6,7]. Nowadays, most
of researches have tended to focus on the activities of saikosaponins, which have been
CONTACT shao-Jiang song
songsj99@163.com; chun-fu Wu
wucf@syphu.edu.cn
the supplemental data for this article is available online at http://dx.doi.org/10.1080/10286020.2016.1234456.
© 2016 informa uK limited, trading as taylor & francis Group

2
D.-Q. LI ET AL.
Figure 1. structures of compounds 1–9.
reported as the major biologically active components with antiviral, anticancer, anti-inflam-
matory activities, and so on [3–6]. However, there is relatively little information pertaining
to isolation and pharmacological activities of the lignans from B. chinense.
As part of a program to search for more constituents with potential antioxidant and antitumor
activities, a new 8-O-4′ neolignan glucoside saikolignanoside A (Figure 1), together with eight
known lignans (7S, 8R)-9′-methoxyl-dehydrodi-coniferyl alcohol 4-O-β-D-glucopyranoside
(2) [8], acutissimalignan B (3) [9], salicifoline (4) [10], (+)-nortrachelogenin (5) [11], guamaro-
lin (6) [12], (+)-pinoresinol (7) [13], pluviatolide (8) [14], and 2-(3″-methoxy-4″-hydroxyben-
zyl)-3-(3′-methoxy-4′- hydroxylbenzyl)-γ-butyrolactone (9) [15] were isolated from the roots
of B. chinense. Herein, we describe the isolation, structural elucidation of the new compound
on the basis of the spectroscopic analysis, including 1D and 2D NMR, HRESIMS, CD, and the
hydrolytic reaction. e antioxidant and cytotoxic activities of the nine lignans were also tested.
2. Results and discussion
Compound 1 was obtained as yellow oil and it showed a quasi-molecular ion peak at m/z
575.2099 [M + Na]+ in the positive HRESIMS, indicating a molecular formula of C27H36O12
with 10 degrees of unsaturation. e strong IR absorption bands indicated the presence

JoURNAL oF ASIAN NATURAL PRoDUCTS RESEARCH
3
Table 1. 1h nMr and 13c nMr spectroscopic data for compound 1.
δH (600 MHz)a
δC (150 MHz)a
Position
1
2
3
4
5
6
7
8
9
1′
2′
3′
4′
5′
6′
7′
132.6
111.2
146.9
145.5
114.7
119.0
70.6
7.01 (1h, o)
6.68 (1h, d, J = 8.2 hz)
6.82 (1h, dd, J = 8.2, 1.6 hz)
4.83 (1h, d, J = 5.2 hz)
4.44 (1h, dt, J = 11.0, 5.2 hz)
82.3
67.8
3.96 (1h, dd, J = 11.0, 3.2 hz)3.22 (1h, dd, J = 11.0, 3.2 hz)
131.5
109.9
149.6
147.8
115.6
119.4
129.7
124.3
72.3
7.07 (1h, d, J = 1.8 hz)
7.01 (1h, o)
6.89 (1h, dd, J = 8.4, 1.8 hz)
6.51 (1h, d, J = 15.9 hz)
6.22 (1h, dt, J = 15.9, 5.3 hz)
4.01 (2h, dd, J = 5.3, 1.4 hz)
4.05 (1h, d, J = 7.8 hz)
2.93–2.97 (1h, m)
3.03 (1h, o)
8′
9′
1″
2″
103.4
73.4
76.8
3″
4″
5″
6″
3-och3
3′-och3
9′-och3
3.02 (1h, o)
69.9
76.6
60.9
55.4
55.6
57.2
3.08–3.11 (1h, m)
3.61–3.64 (1h, m)3.40 (1h, o)
3.73 (3h, s)
3.79 (3h, s)
3.26 (3h, s)
note: nMr spectroscopic data were recorded in dMso-d6 at 600 Mhz (1h nMr) and 150 Mhz (13c nMr).
aassignments are based on 1h, 13c nMr, hMBc, and hsQc experiments; o: the abbreviation for overlapped.
of hydroxyl group (3426 cm−1) and aromatic rings (1639, 1461 and 1384 cm−1). 1H NMR
spectrum (Table 1) of 1 displayed signals of a trans double bond [δH 6.51 (1H, d, J = 15.9 Hz,
H-7′), 6.22 (1H, dt, J = 15.9, 5.3 Hz, H-8′)], two sets of oxygenated methylene protons
[δH 3.96 (1H, dd, J = 11.0, 3.2 Hz, H-9a), 3.22 (1H, dd, J = 11.0, 3.2 Hz, H-9b); 4.01(2H, dd,
J = 5.3, 1.4 Hz, H-9′)], one anomeric proton [δH 4.05 (1H, d, J = 7.8 Hz, H-1″)], two separate
ABX proton signals [δH 7.01 (1H, o, H-2), 6.82 (1H, dd, J = 8.2, 1.6 Hz, H-6), 6.68 (1H, d,
J = 8.2 Hz, H-5) and 7.07 (1H, d, J = 1.8 Hz, H-2′), 7.01 (1H, o, H-5′), 6.89 (1H, dd, J = 8.4,
1.8 Hz, H-6′)], together with three methoxy groups at δH 3.79 (3H, s), 3.73 (3H, s) and 3.26
(3H, s). e functional group signals appearing in the 13C NMR spectrum also included a
double bond, two aromatic rings, and three methoxy groups. e HMBC correlations from
H-7 at δH 4.83 (1H, d, J = 5.2 Hz) to C-1 at δC 132.6, C-6 at δC 119.0, C-2 at δC 111.2, C-8
at δC 82.3 and C-9 at δC 67.8, as well as from H-7′ at δH 6.51 (1H, d, J = 15.9 Hz) to C-2′
at δC 109.9 and C-6′ at δC 119.4, evidenced the linkage positions of two phenyl propanoid
units (Figure 2). e NOESY correlation between H-8 at δH 4.44 (1H, dt, J = 11.0, 5.2 Hz)
and H-5′ at δH 7.01 (1H, o) was also observed. In addition, with the help of 1H-1H COSY
spectrum (Figure 2), the correlations between H-7 at δH 4.83 (1H, d, J = 5.2 Hz) and H-8
at δH 4.44 (1H, dt, J = 11.0, 5.2 Hz), between H-8 at δH 4.44 (1H, dt, J = 11.0, 5.2 Hz) and
H-9 at δH 3.22 (1H, dd, J = 11.0, 3.2 Hz) established that compound 1 had an 8-O-4′ neol-
ignan system. Two methoxy groups were assigned to C-3 and C-3′ positions by the HMBC
correlations from δH 3.73 (3H, s) and 3.79 (3H, s) to the carbons at δC 146.9 (C-3) and δC
149.6 (C-3′), respectively. In addition, a methoxy group was attached to C-9′ by the HMBC
correlations between δH 3.26 (3H, s) and δC 72.3 (C-9′) (Figure 2).

4
D.-Q. LI ET AL.
Figure 2. Key hMBc and 1h-1h cosY correlations of compound 1.
e 1H NMR and 13C NMR spectra also displayed a set of sugar moiety signals [δH 4.05
(1H, d, J = 7.8 Hz, H-1″), 3.61–3.64 (1H, m, H-6a″), 3.40 (1H, o, H-6b″), 3.08–3.11 (1H,
m, H-5″), 3.03 (1H, o, H-3″), 3.02 (1H, o, H-4″) and 2.93–2.97 (1H, m, H-2″), as well as δC
103.4 (C-1″), 60.9 (C-6″), 76.6 (C-5″), 76.8 (C-3″), 69.9 (C-4″), and 73.4 (C-2″)]. Initially,
we assigned the sugar as glucose by comparison of its data with literature. To determine
the absolute configuration, compound 1 was subjected to acidic hydrolysis, followed by GC
analysis in comparison with authentic D-glucose [tR (D-glucose) = 13.83 min]. e results
confirmed the presence of D-glucose moiety in 1. In addition, the β-oriented anomeric
configuration was determined based on the JH–H values (7.8 Hz). e linkage position of the
glucosyl group was assigned by HMBC correlations between the anomeric proton at δH 4.05
(1H, d, J = 7.8 Hz, H-1″) and C-9 at δC 67.8 (Figure 2). Detailed analysis of the 2D NMR
of 1 (Figure 2), along with comparison of the NMR data revealed the similar structure of
1 with those of the debilignanoside [16]. However, one methoxy group at C-9′ (δC 72.3) in
compound 1 appeared instead of a hydroxyl group for debilignanoside. e stereochemistry
of 1 was determined by a comprehensive analysis of the NMR and CD data. e ∆δC8-C7
values (11.7 ppm) of 1 were smaller than those of the threo isomer (13.4 or 13.0 ppm) in
the literatures and suggested a 7, 8-erythro relative configuration [16,17]. e CD spectrum
showed a negative Cotton effect at 248 nm, indicating 8R configuration for compound 1. So,
it was found that the new compound had 7S, 8R-configuration [18,19]. us, the structure
of 1 was determined and named saikolignanoside A.
Reactive oxygen species (ROS) increase the chance of occurrence of cancer, atherosclerosis,
neurodegenerative diseases, and inflammation [20]. Several studies demonstrated that
lignans can decrease the incidence of oxidative stress and associated diseases [21,22].
Compounds 1–9 were evaluated for antioxidant effects using the DPPH and ABTS assays
(Table 2). Trolox served as a positive control, with IC50 values of 5.70 and 12.21 μM,
respectively. As shown in Table 2, all compounds were inactive (IC50 > 200 μM) in the
DPPH assay. But in the ABTS assay, compounds 1–2, 5, 7, and 9 display significant ABTS
radical-scavenging activities, whereas the other compounds showed moderate activities
comparable with that of the standard compound trolox (IC50 = 5.70 μM).
It has been reported that antioxidants play an important role in protecting the body
from oxidative stress by scavenging free radicals and preventing cancer invasion [23]. In
order to evaluate the potential cytotoxic activities, all isolated lignans were tested for their
cytotoxic activities against the five selected human tumor cell lines by the MTT method
(Table 3). e results obtained are expressed as IC50 values in μM. 5-Fluorouracil was used

JoURNAL oF ASIAN NATURAL PRoDUCTS RESEARCH
5
Table 2. free radical scavenging activity of compounds 1–9.
Compounds
DPPH (IC50, μM)
ABTS (IC50, μM)
1
>200
>200
>200
>200
>200
>200
>200
88.52
>200
12.21
8.34
15.24
32.70
25.62
15.13
19.87
13.43
23.20
10.62
5.70
2
3
4
5
6
7
8
9
troloxa
atrolox was used as the positive control.
Table 3. cytotoxic activities of compounds 1–9 in vitro (ic50, μM).
IC50 (μM)a Cell line
Compounds
A549
HepG2
U251
Bcap-37
MCF-7
1
20.11
>80
30.77
>80
19.61
20.11
29.35
>80
17.05
34.27
32.021
>80
>80
29.18
17.62
32.02
62.35
>80
15.14
49.72
>80
>80
>80
>80
39.10
>80
>80
>80
23.92
48.60
>80
>80
>80
>80
51.62
>80
>80
>80
50.32
47.09
50.63
>80
>80
2
3
4
>80
5
45.64
50.63
75.32
>80
25.34
42.78
6
7
8
9
5-fub
aic50 (μM) represents means sd of three independent replicates. the ic50 greater than 80 μM was considered to indicate
no cytotoxicity.
b5-fu (5-fluorouracil) was used as positive control.
as positive control. In Table 3, compounds 5, 9 possessed stronger cytotoxic activities against
all the selected tumor cell lines. In addition, compounds 1, 6 possessed stronger cytotoxic
activities against the A549 and HepG2 human tumor cell lines.
In this study, antioxidant and cytotoxic effects of the lignans isolated from B. chinense
were reported for the first time. ese findings were valuable for further investigation and
optimization of the roots of B. chinense. On the basis of these promising results, compounds
5 and 9 which exhibited potent antioxidant and antitumor activities deserve further explo-
ration for the deeper investigations into its mechanism of action, as well as their anticancer
effects in vivo.
3. Experimental
3.1. General experimental procedures
Optical rotations were measured on a JASCO DIP-370 digital polarimeter (Jasco Co., Tokyo,
Japan). e UV spectra were obtained on a Shimadzu UV-1700 spectrometer (Shimadzu
Corporation, Kyoto, Japan). IR spectra were recorded on a Bruker IFS-55 spectrometer
(Bruker Co., Karlsruhe, Germany). HR-ESIMS data were measured on an Agilent G6520
Q-TOF LC/MS spectrometer (Bruker Co., Karlsruhe, Germany). CD spectra were obtained
on MOS 450 detector in the 200–400 nm wavelength range (Bio-Logic Co., Claix, France).

6
D.-Q. LI ET AL.
NMR spectra were recorded on Bruker AV-400 and AV-600 spectrometers (Bruker Co.,
Billerica, MA, U.S.A) with TMS as internal standard. An Agilent 1100 HPLC (Agilent,
Santa Clara, CA, U.S.A) was used for isolation and purification with YMC C-18 semi-pre-
parative HPLC column (5 μm, 250 × 10 mm; YMC Co. Ltd., Kyoto, Japan). GC was carried
out using an Agilent 7890A gas chromatograph (Agilent Technologies, Inc., Santa Clara,
CA, U.S.A) equipped with an HP-5 capillary column (30 m × 0.32 mm × 0.25 μm; Agilent
Technologies, Inc., Santa Clara, CA, U.S.A). Column chromatography (CC) was performed
on macroporous adsorption resin D101 (Cangzhou Bon Adsorber Technology Co., Ltd.,
Cangzhou, China), Silica gel (200–300 mesh, Qingdao Marine Chemical Factory, Qingdao,
China), ODS silica gel (50 μm, YMC Co. Ltd., Kyoto, Japan) and Sephadex LH-20 (25–100 m,
Green Herbs Science and Technology Development Co., Ltd., Beijing, China). e fractions
were monitored by TLC (SiO2 GF254; Qingdao Marine Chemical Inc., Qingdao, China).
DPPH and ABTS assays were performed on Varioskan Flash Multimode Reader (ermo
Scientific, Waltham, MA, U.S.A).
3.2. Plant material
e roots of Bupleurum chinense were purchased from Liaoning Tongren Pharmaceutical
Co. Ltd (Shenyang, China), and identified by professor Jin-Cai Lu (Department of
Natural Products Chemistry, Shenyang Pharmaceutical University). A voucher specimen
(No.1012002) has been preserved in the Herbarium of Shenyang Pharmaceutical University.
3.3. Extraction and isolation
e dried plant material (9.5 kg) was extracted by homogenate extraction with 75% etha-
nol. Evaporation of the ethanol extract afforded 800.0 g dark gummy residue. e residue
(800.0 g) was chromatographed on silica gel (200–300 mesh) with a gradient of CH2Cl2/
MeOH of 100: 1, 50: 1, 30: 1, 20: 1, 10: 1 and 0: 1, to afford five fractions (Fr. 1-5). Fr. 2
(102.0 g) was further separated by silica gel CC eluting with a CH2Cl2/MeOH gradient sys-
tem (v/v = 50: 1, 25: 1, 10: 1, 8: 1, 5: 1 and 0: 1) to yield 6 fractions (Fr. 2-1-Fr. 2-6), Fr. 2-2
(10.4 g) was separated by C18 column chromatography and eluted with MeOH–H2O gradient
(v/v = 30: 70, 60: 40 and 100: 0) to give 5 fractions (Fr. 2-2-1-Fr. 2-2-5). Fr. 2-2-2 (1.5 g)
was purified by semi-preparative HPLC (solvent: 25% CH3CN-H2O) to give 1 (10.3 mg,
tR = 15.4 min) and 2 (14.5 mg, tR = 21.3 min). Fr. 2-2-3 (2.5 g) was chromatographed on
Sephadex LH-20 (MeOH) and further purified by semi-preparative HPLC (solvent: 35%
MeOH–H2O) to yield 3 (11.5 mg, tR = 13.1 min), 9 (15.2 mg, tR = 15.6 min), 5 (10.1 mg,
tR = 21.6 min) and 6 (7.3 mg, tR = 30.6 min). Fr. 2-2-4 (2.3 g) was also chromatographed over
Sephadex LH-20 and eluted with MeOH to give compound 8 (13.1 mg, tR = 15.6 min) and
four subfractions A-D. Compound 7 (14.1 mg, tR = 17.8 min) and compound 4 (11.3 mg,
tR = 24.3 min) was purified from subfraction B by HPLC (solvent: 37% MeOH–H2O).
3.3.1. Saikolignanoside A
Yellow oil,[ꢀ]2D0-17.3 (c 0.1, CH OH); UV (MeOH) λmax (log ε): 281.0 (2.96) nm; IR (KBr) νmax
:
3
3426, 2920, 2850, 1639, 1461, 1384, 1338, 1263 1130, 878 and 619 cm−1; CD (c 0.10 mg/ml,
MeOH): Δε248 nm −8.86; 1H and 13C NMR spectral data, see Table 1; HRESIMS: m/z 575.2099
[M + Na]+ (calcd for C27H36O12Na, 575.2098).

JoURNAL oF ASIAN NATURAL PRoDUCTS RESEARCH
7
3.4. Acid hydrolysis of compound 1
A solution of compound 1 (2.0 mg) was heated in 1 M HCl (2.0 ml) at 95 °C for 4 h in a
sealed ampoule and extracted with EtOAc (3 × 2.0 ml). e aqueous layer was concentrated
to dryness, to yield a residue. is residue was dissolved in pyridine (1.0 ml), and then
L-cysteine methyl ester hydrochloride (2.0 mg) was added to the solution. e mixture
was heated at 60 °C for 2 h, and 0.1 ml N-(trimethylsily)-imidazole (TMSI) was added,
followed by heating at 60 °C for 1 h. Afer drying the solution, the residue was partitioned
between H2O (2 ml) and n-hexane (2 ml). e n-hexane layer was subjected to GC anal-
ysis on Agilent 7890A (HP-5, 30 m × 320 mm × 0.25 μm) with flame ionization detection
under the following conditions: e column temperature was set at 150–280 °C at the rate
of 10 °C/min, and the carrier gas was N2 (1.4 ml/min); the injection temperature was 250 °C
and the injection volume, 1 μl. e absolute configurations of the monosaccharides were
identified to be D-Glu by comparison of the retention times of its Me3Si ethers with those
of standard samples [tR (D-glucose) = 13.83 min].
3.5. Antioxidant assays
3.5.1. DPPH radical scavenging assay
e DPPH (1, 1-diphenyl-2-picrylhydrazyl) radical-scavenging effect was performed as
previously described method with some modifications [24]. Trolox (Purity. 98%; Sigma, Los
Angeles, CA, U.S.A) was used as the positive control. Tests were performed in triplicate. e
samples with different concentrations (0.1–100 μM) in EtOH (100 μl) were added to 0.2 mM
DPPH in EtOH (150 μl). e mixture was placed in a 96-well microplate and incubated
at 37 °C for 30 min. e scavenging activity of samples was estimated by measuring the
absorption of the mixture at 517 nm with a Varioskan Flash (ermo Scientific, Waltham,
MA, U.S.A) and the means of three readings were calculated. DPPH radical scavenging
(capacity) (%) = [1−(S−Sb)/(C−Cb)] × 100%, where S sample is the absorbance of the test
sample, Sb is the blank sample, C is the absorbance of the control, and Cb is the blank control,
respectively. e scavenging activity was expressed as IC50, the concentrations of samples
required for scavenging 50% of DPPH radical in the solution.
3.5.2. ABTS radical scavenging activity assay
e radical scavenging activity of compounds (1–9) was tested using the improved ABTS
decolorization assay with modification [25]. Trolox, a water-soluble analog of vitamin E, was
used as a positive control. ABTS, 2, 2′-azinobis (3-ethyl-benzothiazoline-6-sulfonic acid)
diammonium salt was dissolved in water to a 7 mM concentration. ABTS radical cation
(ABTS•+) was produced by reacting ABTS solution with 2.45 mM K2S2O8 (final concentra-
tion) and allowing the mixture to store in the dark at room temperature for 12–16 h before
use. For analysis, the ABTS•+ solution was diluted with ethanol so that the absorbance at
734 nm was 0.700 0.020. e diluted ABTS•+ solution (150 μl) was mixed with ethanolic
solution (50 μl) of samples at various concentrations, and then the absorbance was detected
at 734 nm afer 20 min. All experiments involving these samples were replicated three times.
ABTS•+ radical scavenging activity of the isolated compounds was calculated by formula
as below: ABTS•+ scavenging activity (%) = [1 − (S−Sb)/(C−Cb)] × 100%, where S, Sb, C
and Cb are the absorbencies of the sample, the blank sample, the control, and the blank
control, respectively.

8
D.-Q. LI ET AL.
3.6. In vitro cytotoxicity assay
All isolated compounds (1–9) were subjected to cytotoxic evaluation against A549, HepG2,
U251, Bcap-37and MCF-7 cell lines employing the MTT (3-(4, 5- dimethylthiazol-2-yl)-2,
5-diphenyltetrazolium bromide) method with 5-Fluorouracil as the positive control [24].
Briefly, cells were seeded into each well of a 96-well cell culture plate and incubated at 37 °C
for 24 h before drug treatment. Compounds (1–9) and 5-fluorouracil were then added to
make various final concentrations (6.25, 12.5, 25, 50, 100 μM). e cells were then exposed
to 20 μl of the test compounds at various concentrations in triplicate and incubated for 48 h.
Afer the incubation, MTT solution (0.5 mg/ml) was added to each well, and the incubation
continued for 4 h at 37 °C. e supernatant was decanted, and DMSO (100 μl/well) was
added to allow formazan solubilization. e level of MTT formazan was determined by
measuring its absorbance at 492 nm with the Spectra Shell reader. e percentage of cell
inhibition was calculated as follows: Cell death (%) = [A492 (control)−A492 (test)]/A492
(control) × 100%. IC50 values were calculated with the LOGIT method.
Disclosure statement
No potential conflict of interest was reported by the authors.
Funding
is research was financially supported by the National Natural Sciences Foundation of China [grant
number 81130071]; the Key Laboratory of Structure-Based Drug Design and Discovery, Ministry of
Education and the Foundation [LT2015027] from the Project of Innovation Team.
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