Fitoterapia 132 (2019) 68–74

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Fitoterapia
journal homepage: www.elsevier.com/locate/fitote

Prenylated flavans from Daphne giraldii and their cytotoxic activities T

a,b a a a c a a
Qian Sun , Xin-Yue Shang , Yu-Xi Wang , Guo-Dong Yao , Fei-Fei Li , Ling-Zhi Li , Yan Zhang ,
⁎
Xiao-Xiao Huanga, Shao-Jiang Songa,
a
School of Traditional Chinese Materia Medica, Key Laboratory of Computational Chemistry-Based Natural Antitumor drug Research & Development, Shenyang
Pharmaceutical University, Shenyang, Liaoning 110016, People's Republic of China
b
Xiamen Institute for Food and Drug Quality Control, Xiamen 361012, People's Republic of China
c
School of Chinese Pharmacy, Beijing University of Chinese Medicine, Beijing 100102, China

A R T I C LE I N FO A B S T R A C T

Keywords: Nine new prenylated flavan compounds with multi-chiral centers including two pairs of epimers were isolated
Daphne giraldii from the stem and root bark of Daphne giraldii. Their structures were established by extensive NMR and HR-
Prenylated flavans ESIMS spectroscopic data analyses. The in vitro cytotoxicity experiments indicated that compound 6 showed the
Apoptosis most significant cytotoxicity against Hep3B cells, with an IC50 value of 9.83 μM. Hoechst 33258 and Annexin V-
FITC/PI staining suggested that 6 could induce apoptosis of Hep3B cells in a concentration-dependent manner.
Further mechanism study indicated that the apoptosis was associated with the up-regulations of Bax, cl-PARP
and a decrease in Bcl-2 expression.

1. Introduction 2. Materials and methods

Daphne giraldii Nitsche (Thymelaeaceae) is mainly distributed in the
northwest region of China [1]. The stem and root bark of this herb,
commonly known as ‘ZuShima’ have been used as an antirheumatic and
antalgic agent in traditional Chinese medicine [2]. Previous phyto-
chemical studies have led to the isolation of a number of bioactive
constituents, including coumarins [3], flavonoids [4], diterpenes [5],
and lignans [6], some of which exhibit cytotoxic [7], anti-inflammatory
[8], antifertility [9], antimalarial [10], and sedativehypnotic activities
[11]. Among them, prenylated flavans are a kind of attractive con-
stituents discovered in recent years. They not only have variable che-
mical structures derived from the cyclization between the prenyl and
adjacent hydroxyl moiety into furan or pynan rings, but also possess
marked cytotoxicity with mechanisms involving the induction of
apoptosis [12], cell cycle arrest [13], autophagy [14] and NF-κB in-
hibition [15]. As our continuous investigation for cytotoxic pyenylated
flavans from the D. giraldii, materials in the remaining fractions were
further fractionated using column chromatography as well as HPLC to
afford nine new flavans (1-9) (Fig. 1). Herein, we reported the isolation,
structural elucidation and in vitro cytotoxicity of these compounds.
Moreover, compound 6 was evaluated for further apoptosis of Hep3B
cells.
2.1. General experimental procedures
UV and IR (KBr) spectra were obtained on an UV-1700 spectro-
photometer (SHIMADZU, Japan) and a Bruker IFS 55 spectro-
photometer (Bruker, Germany), respectively. Optical rotations were
acquired on an AUTOPOL IV automatic polarimeter (Rudolph Research
Analytical, USA). CD spectra were recorded on a MOS-450 spectrometer
(Bio-Logic Science, France). NMR experiments were carried out on
Bruker ARX-300 and AV-600 NMR instruments. HRESIMS analyses
were performed on a Bruker Micro Q-TOF spectrometer. Silica gel
(100−200 or 200−300 mesh; Qingdao Marine Chemical Inc. China),
Sephadex LH-20 gel (GE Healthcare, Sweden), ODS (60–80 μm; Merck,
Germany) and MCI gel (CHP20P, 75−150 μm; Mitsubishi Chemical
Industries Ltd. Japan) were used for normal column chromatography.
Pre-coated silica gel GF254 plates (Yantai Zifu Chemical Group Co.
China) were used for analytical TLC. Purification of the compounds was
conducted on a Shimadzu LC-6A equipped with a SPD-20A UV/VIS
detector using an YMC Pack ODS-A column (250 × 10 mm, 5 μm).
2.2. Plant material
The stem and root bark of D. giraldii were obtained from Anguo
herbal medicine market (HeBei, China), in May 2012, and identified by

⁎
Corresponding author.
E-mail address: songsj99@163.com (S.-J. Song).

https://doi.org/10.1016/j.fitote.2018.11.011
Received 10 September 2018; Received in revised form 16 November 2018; Accepted 24 November 2018
Available online 27 November 2018
0367-326X/ © 2018 Elsevier B.V. All rights reserved.
Q. Sun et al.
Fig. 1. Chemical structures of compounds 1–9.
Prof. Jin-Cai. Lu, Shenyang Pharmaceutical University. A voucher
specimen (DG-20120515) has been deposited at the Department of
Natural Products Chemistry, Shenyang Pharmaceutical University.
2.3. Extraction and isolation
The air-dried stem and root bark of D. giraldii were extracted three
times with 95% EtOH at room temperature. The solvent of the extrac-
tion was removed under reduced pressure to yield the crude extract
(986.0 g). Then it was subjected to silica gel column chromatography
by using CH2Cl2–MeOH (100:1, 30:1, 15:1, 0:100, v/v) to give fractions
A–D. The fraction B (280.0 g) was chromatographed on repeated silica
gel by eluted with PE–EtOAc (50:1, 20:1, 10:1, 0:100, v/v), and the
third fraction B3 (65.3 g) was further separated by MCI gel
(MeOH–H2O, 40:60, 60:40, 80:20, 100:0, v/v) to obtain four fractions.
Fraction B3c (24.0 g) was chromatographed over ODS (MeOH–H2O,
50:50, 60:40, 70:30, 80:20, v/v) to yield 12 parts named B3c-1–B3c-12.
Fr B3c-2 (0.8 g) was purified through preparative HPLC (MeCN–H2O,
55:45, v/v) to give compound 7 (2.5 mg, tR=25.6 min) and 8 (4.8 mg,
tR=27.0 min). Fr B3c-3 (0.8 g) was purified by HPLC ((MeCN–H2O,
45:55, v/v) to yield 9 (3.8 mg, tR=15.1 min). Sequential separation of
B3c-8 (2.7 g) over silica gel (PE–EtOAc, 20:1, 10:1, 5:1, 3:1, 1:1, v/v)
produced ten fractions (B3c-3-1–B3c-3-10). Fr B3c-3-6 (74.7 mg) was
separated by preparative HPLC (MeCN–H2O, 35:65, v/v) to afford
compound 1 (11.2 mg, tR=17.2 min), 2 (15.0 mg, tR=18.6 min), 4 (4.0
mg, tR=19.5 min) and 5 (2.4 mg, tR=20.3 min). A portion of fraction
B3c-3-7 (174 mg) was fractionated by HPLC (MeCN–H2O, 40:60, v/v)
to give 3 (4.8 mg, tR=10.6 min). Similarly, HPLC purification was
performed on fraction B3c-3-8 (119.0 mg) to afford compound 6
(2.5mg, tR=10.2 min).
69
Fitoterapia 132 (2019) 68–74
2.4. Characterization of compounds 1-9
Daphnegiralin E (1). Yellow solid (MeOH); [α]20 D-10.0 (c 0.10,
MeOH); CD (MeOH) nm (Δε) 284 (-0.46); UV (MeOH) λmax nm (log ε)
229 (3.3), 274 (2.6), 284 (2.6); IR (KBr) vmax 3406, 2972, 2927, 1622,
1509, 1460 cm−1; 1H NMR and 13C NMR data, see Tables 1 and 2;
HRESIMS m/z 431.1828 [M+Na]+ (calcd for C25H28O5Na 431.1829).
Daphnegiralin F (2). Yellow solid (MeOH); [α]20 D+11.4 (c 0.10,
MeOH); CD (MeOH) nm (Δε) 288 (-0.72); UV (MeOH) λmax nm (log ε)
277 (2.3); IR (KBr) vmax 3420, 2972, 2927, 1623, 1509, 1450 cm−1; 1H
NMR and 13C NMR data, see Tables 1 and 2; HRESIMS m/z 431.1824
[M+Na]+ (calcd for C25H28O5Na 431.1829).
Daphnegiralin G (3). Yellow solid (MeOH); [α]20 D-8.8 (c 0.06,
MeOH); CD (MeOH) nm (Δε) 282 (-0.61); UV (MeOH) λmax nm (log ε)
229 (3.3), 274 (2.6), 284 (2.6); IR (KBr) vmax 3406, 2972, 2927, 1622,
1509, 1460 cm−1; 1H NMR and 13C NMR data, see Tables 1 and 2;
HRESIMS m/z 433.1986 [M+Na]+ (calcd for C25H30O5Na 433.1985).
Daphnegiralin I (4). Yellow solid (MeOH); [α]20 D-13.8 (c 0.08,
MeOH); CD (MeOH) nm (Δε) 282 (-0.93); UV (MeOH) λmax nm (log ε)
284 (2.8); IR (KBr) vmax 3382, 2973, 2926, 1621, 1509, 1479 cm−1; 1H
NMR and 13C NMR data, see Tables 1 and 2; HRESIMS m/z 417.2036
[M+Na]+ (calcd for C25H30O4Na 417.2036).
Daphnegiralin G (5). Yellow solid (MeOH); [α]20 D+5.6 (c 0.05,
MeOH); CD (MeOH) nm (Δε) 283 (-1.04); UV (MeOH) λmax nm (log ε)
283 (2.8); IR (KBr) vmax 3393, 2973, 2927, 1621, 1509, 1479 cm−1; 1H
NMR and 13C NMR data, see Tables 1 and 2; HRESIMS m/z 395.2216
[M+H]+ (calcd for C25H31O4 395.2217).
Daphnegiralin K (6). Yellow solid (MeOH); [α]20 D-18.5 (c 0.05,
MeOH); CD (MeOH) nm (Δε) 287 (-0.56); UV (MeOH) λmax nm (log ε)
231 (3.1), 277 (2.4), 285 (2.4); IR (KBr) vmax 3427, 2974, 2926, 1623,
1509, 1461 cm−1; 1H NMR and 13C NMR data, see Tables 1 and 2;
HRESIMS m/z 431.1830 [M+Na]+ (calcd for C25H28O5Na 431.1829).
Daphnegiralin L (7). Yellow solid (MeOH); [α]20 D-5.0 (c 0.07,
Q. Sun et al. Fitoterapia 132 (2019) 68–74

Table 1 Table 3
13 1
C NMR spectroscopic data for compounds 1-9. H NMR spectroscopic data for compounds 7-9 (DMSO-d6 at 600 MHz).
No 1a 2a 3a 4a 5a 6b 7c 8c 9c No 7 8 9

2 76.5 76.2 76.0 79.3 79.4 76.7 74.4 74.3 76.8 2 4.96 dd (10.6, 1.2) 4.98 dd (10.5, 1.6) 4.97 dd (10.0, 1.9)
3 30.2 30.5 29.9 31.5 31.5 28.5 29.6 29.5 29.1 3 2.00 m, 1.82 m 2.01 m, 1.86 m 2.07 m, 1.96 m
4 26.3 26.2 26.0 25.5 25.5 24.9 24.8 24.6 23.9 4 2.81 m, 2.67 m 2.81 m, 2.65 m 2.81 m, 2.61 m
5 131.0 131.0 131.0 130.9 130.9 130.4 129.9 129.8 129.8 5 6.87 d (8.2) 6.86 d (8.2) 6.85 d (8.2)
6 109.2 109.3 109.1 109.0 109.0 108.1 108.0 108.0 108.0 6 6.28 dd (8.2, 2.3) 6.28 dd (8.2, 2.4) 6.28 dd (8.2, 2.0)
7 157.5 157.6 157.5 157.6 157.6 155.0 156.4 156.4 156.5 8 6.16 d (2.3) 6.17 d (2.4) 6.19 d (2.0)
8 104.1 104.1 104.0 104.0 104.0 103.6 102.8 102.8 102.8 2′ — — 7.44 d (2.0)
9 157.3 157.3 157.4 157.2 157.2 156.0 155.8 155.9 155.5 3′ — — —
10 114.4 114.3 114.4 114.3 114.3 114.2 112.2 112.1 112.1 4′ — — —
1′ 133.9 133.9 130.9 135.3 135.3 129.2 131.2 132.1 133.6 5′ — — 6.84 d (8.2)
2′ 126.6 125.9 118.6 121.2 121.2 126.8 124.3 128.0 123.9 6′ 6.97 s 6.93 s 7.28 dd (8.2, 2.0)
3′ 144.8 144.8 142.6 128.0 128.0 147.3 142.3 138.3 126.8 1″ 3.32 m 3.38 m —
4′ 140.9 140.8 144.8 158.8 158.8 136.7 139.5 148.4 160.2 2″ 5.04 t (6.9) 5.05 t (7.2) 4.25 d (2.8)
5′ 121.2 121.3 123.7 123.6 123.6 122.0 123.3 124.7 109.3 3″ — — 4.98 d (2.8)
6′ 116.4 116.0 120.6 126.9 126.9 116.7 115.6 113.9 128.6 4″ 1.61 s 1.62 s —
1″ 33.8 33.7 24.3 24.2 5″ 1.65 s 1.65 s 1.00 s
2″ 77.1 77.1 75.1 90.1 90.1 90.3 123.6 123.4 93.8 6″ — — 1.19 s
3″ 149.3 149.1 33.8 31.7 31.7 30.4 129.9 130.0 80.6 1″′ — — —
4″ 110.8 111.0 20.3 72.6 72.6 72.1 25.5 25.4 69.6 2″′ 3.36 o 4.23 d (2.5) —
5″ 18.4 18.2 26.6 24.7 24.6 24.0 17.8 17.7 24.2 3″′ 4.29 t (6.0) 4.95 d (2.5) —
6″ 27.0 25.9 25.9 26.2 26.5 4″′ — — —
1″′ 37.9 29.2 29.2 5″′ 1.12 s 0.98 s —
2″′ 77.7 77.7 76.9 123.5 123.5 76.7 75.0 93.9 6″′ 1.40 s 1.21 s —
3″′ 131.8 131.8 148.8 133.2 133.2 130.7 68.1 81.4 3″′-OCH3 — 3.30 s 3.34 s
4″′ 123.4 123.4 111.2 17.9 17.9 122.5 78.9 69.7
5″′ 27.9 27.9 18.1 25.9 25.9 28.0 19.2 24.0 o: The abbreviation for overlapped.
6″′ 28.0 28.0 28.2 26.7 26.7
3″′-OCH3 54.9 55.1
see Tables 1 and 3; HRESIMS m/z 463.2090 [M+Na]+ (calcd for
a
Data were measured in methanol-d4 at 150 MHz. C26H32O6Na 463.2091).
b
Data were measured in CDCl3 at 150 MHz. Daphnegiralin N (9). Yellow solid (MeOH); [α]20 D-6.0 (c 0.08,
c
Data were measured in DMSO-d6 at 150 MHz. MeOH); CD (MeOH) nm (Δε) 282 (-0.59); 1H NMR and 13C NMR data,
see Tables 1 and 3; HRESIMS m/z 379.1510 [M+Na]+ (calcd for
MeOH); CD (MeOH) nm (Δε) 282 (-0.75); 1H NMR and 13C NMR data, C21H24O5Na 379.1516).
see Tables 1 and 3; HRESIMS m/z 449.1936 [M+Na]+ (calcd for
C25H30O6Na 449.1935). 2.5. Cell culture
Daphnegiralin M (8). Yellow solid (MeOH); [α]20 D-6.2 (c 0.06,
MeOH); CD (MeOH) nm (Δε) 284 (-0.41); 1H NMR and 13C NMR data, Hep3B, Bcap37, A549, U251, MCF-7, HepG2 cell lines were

Table 2
1
H NMR spectroscopic data for compounds 1-6.
No 1a 2b 3a 4a 5a 6c

2 5.13 dd (10.8, 2.0) 5.16 dd (10.7, 1.8) 5.03 dd (10.0, 2.2) 4.88 o 4.87 o 4.89 m
3 2.17 m, 2.05 m 2.10 m, 2.03 m 2.08 m, 2.02 m 2.06 m, 1.97 m 2.07 m, 1.97 m 2.09 m, 2.09 m
4 2.92 m, 2.73 m 2.91 m, 2.74 m 2.89 m, 2.72 m 2.82 m, 2.64 m 2.82 m, 2.63 m 2.89 m, 2.76 m
5 6.88 d (8.4) 6.89, d (8.4) 6.88 d (8.2) 6.85 d (8.2) 6.84 d (8.2) 6.93 d (8.1)
6 6.32 dd (8.4, 2.4) 6.33 dd (8.4, 2.4) 6.32 dd (8.2, 2.4) 6.31 dd (8.2, 2.4) 6.31 dd (8.2, 2.4) 6.38 o
8 6.23 d (2.4) 6.24 d (2.4) 6.24 d (2.4) 6.24 d (2.4) 6.24 d (2.4) 6.37 o
2′ — — — 7.03 brs 7.03 brs —
3′ — — — — — —
4′ — — — — — —
5′ — — — — — —
6′ 6.70 s 6.71 s 6.77 s 6.91 brs 6.91 brs 6.56 s
1″ 2.98 dd (12.8, 8.5) 3.07 dd (13.6, 4.8) — — — —
2.88 o 2.81 dd (13.6, 8.0)
2″ 4.37 dd (8.5, 4.3) 4.37 dd (8.0, 4.8) — 4.58 t (8.4) 4.57 t (8.4) 4.67 t (9.0)
3″ — — 1.84 t (6.8) 3.18 m, 3.15 m 3.18 m, 3.15 m 3.19 m, 3.17 m
4″ 4.91 brs, 4.76 o 4.88 o, 4.76 brs 2.85 o, 2.74 o — — —
5″ 1.77 s 1.79 s 1.35 s 1.22 s 1.22 s 1.20 s
6″ — — 1.37 s 1.25 s 1.25 s 1.34 s
1″′ — — 2.91 o, 2.75 o 3.28 m, 3.25 m 3.28 m, 3.25 m —
2″′ — — 4.34 dd (8.0, 5.2) 5.29 m 5.29 m —
3″′ 5.66 d (9.8) 5.67 d (9.9) — — — 5.58 d (9.8)
4″′ 6.34 d (9.8) 6.34 d (9.9) 4.86 brs, 4.75 o 1.71 s 1.71 s 6.30 d (9.8)
5″′ 1.44 s 1.44 s 1.77 s 1.71 s 1.71 s 1.46 s
6″′ 1.45 s 1.44 s — — — 1.47 s

o: The abbreviation for overlapped.

a
Recorded in methanol-d4 and 300 MHz.
b
Recorded in methanol-d4 and 600 MHz.
c
Recorded in CDCl3 and 300 MHz.

70
Q. Sun et al.
Fig. 2. Key HMBC and COSY correlations of compounds 1-9.
obtained from the America Type Culture Collection (USA). The cells
were cultured in Dulbecco's modified eagle medium (DMEM) (Hyclone,
USA) supplemented with 10% fetal bovine serum (Biological Industries,
Israel) in 37 °C, 5% CO2 incubator.
2.6. Growth inhibition assay
Growth inhibition was detected using the MTT (Amersco, USA)
assay with compounds in 96-well plates at a density of 5×103 cells per
well in medium and allowed to attach for about 12 h. Cells were treated
with a range of concentrations (6.25, 12.5, 25, 50, 100 μM). After 48 h,
20 μL MTT was added to the wells, and cells were incubated for another
4 h at 37 °C. The medium was removed and DMSO (150 μL/well) was
added to each well. Cell viability was measured at 490 nm. All tests
needed three separate experiments.
2.7. Hoechst 33258 staining
Hep3B cells were seeded at 5×104 cells per well into 24-well plates
for 12 h prior to the experiment. After treatment with different con-
centrations of 6 for 48 h, the suspension was removed, then the cells
were fixed and washed with PBS for three times, then stained with
Hoechst 33258 with the final concentration of 10 μg/mL, incubated at
room temperature in the dark for 25 min according to the manu-
facturer’s instructions. Finally, apoptotic morphological changes in the
nucleus were investigated by florescence microscope (Olympus, Japan).
2.8. Annexin V-PITC/PI staining
Hep3B cells (2×105 cells per well) were seeded in 6-well plates.
After 24 h, cells were treated with 6 (5, 10, 20 μM) for 48 h. Then, both
adherent and floating cells were harvested, centrifuged and washed
71
Fitoterapia 132 (2019) 68–74
with cold PBS. Finally, cells were resuspended in binding buffer and
stained with Annexin V-FITC and propidium iodide (PI) (bimake,
Houston, TX, USA) for 15 min at room temperature in the darkness. The
samples were then analyzed using flow cytometer and quantified with
Flow Jo 7.6.1.
2.9. Western blot analysis
Hep3B cells were seeded into 6-well plates for 24 h and treated with
6 (5, 10, 20 μM) for 48 h. The cells were collected, and lysed in RIPA
lysis buffer (Beyotime, Haimen, China) consisting with 1 mM PMSF and
lyse at 4 °C for 40 min. After 16,000 ×g centrifugation for 15 min, the
protein contents of supernatant are determined by the BCA assay kit
(Beyotime, Haimen, China). The cell extracts were added sample
loading buffer and denatured at 100 °C for 10 min. Equal amount of
protein (30 μg) were separated by a 10% SDS-PAGE gel and transferred
onto PVDF membranes (0.2 μm, Millipore) using an electroblotting
apparatus (Bio-Rad). Then the membranes were blocked with 5% milk
in PBS-T (PBS containing 0.1% Tween 20) for 2 h at room temperature
and probed with primary antibodies at 4 °C overnight. After washed
three times with PBS-T, the membranes were incubated with horse-
radish peroxidase (HRP)-conjugated secondary antibody (1:5000) at
room temperature for 2 h, and then washed again. Proteins are detected
using polyclonal antibodies and visualized using anti-rabbit or anti-
mouse IgG conjugated with peroxidase (HRP) ECL (ThermoFisher,
Waltham, MA) Detection Reagent as the HRP substrate. ImageJ soft-
ware was used to quantify the intensity of the bands.
2.10. Statistical analysis
The results were confirmed in at least three independent experi-
ments. Error bars express the standard deviation (S.D.). Statistical
Q. Sun et al. Fitoterapia 132 (2019) 68–74

Table 4
Cytotoxicity (IC50) for 1-9 against Bcap37, MCF-7, U251, A549, HepG2 and Hep3B cellsa.
Compound Bcap37 MCF-7 U251 A549 HepG2 Hep3B

1 27.08 ± 6.05 23.15 ± 2.81 41.65 ± 2.94 18.77 ± 1.01 21.35 ± 1.17 26.26 ± 0.86
2 > 100 > 100 > 100 22.36 ± 1.65 55.7 ± 4.25 49.89 ± 2.86
3 > 100 > 100 > 100 > 100 44.49 ± 3.26 56.78 ± 4.03
4 > 100 > 100 > 100 79.08 ± 5.11 30.48 ± 2.47 22.78 ± 2.08
5 > 100 > 100 > 100 > 100 > 100 59.82 ± 4.04
6 > 100 91.66 ± 5.76 > 100 > 100 49.99 ± 3.39 9.83 ± 2.85
7 > 100 > 100 > 100 > 100 > 100 > 100
8 > 100 > 100 > 100 > 100 > 100 49.33 ± 4.16
9 > 100 > 100 > 100 > 100 > 100 54.46 ± 4.55
5-Fu 47.09 ± 5.24 42.78 ± 5.19 48.68 ± 6.60 34.27 ± 1.11 49.72 ± 5.74 10.53 ± 1.88

a
Results are expressed as IC50 means ± SD in μM. The experiments were performed three times.

configurations at C-2″ and these two new compounds were named

Fig. 3. Compound 6 inhibited cell viability of Hep3B cells. Cells were treated
with the indicated concentrations of 6 for 48 h, and the cell viabilities were
determined using the MTT assay. The values were expressed as the mean ± SD
of three individual experiments.
analysis of data is made by the student's t-test (GraphPad Prism 6
software). P < 0.05 is considered statistically significant.
3. Results and discussion
Compound 1 was obtained as a yellow solid and the NMR data as
well as the HRESIMS ion peak at m/z 431.1828 [M+Na]+ (calcd
431.1829) suggested a molecular formula of C25H28O5 for 1. The 1H
NMR spectrum showed typical signals of a flavan skeleton at [δH 5.13
(1H, dd, J=10.8, 2.0 Hz, H-2), 2.17, 2.05 (each 1H, m, H-3), 2.92, 2.73
(each 1H, m, H-4)] and one ABX spin system at δH [6.88 (1H, d, J=8.4
Hz, H-5), 6.32 (1H, dd, J=8.4, 2.4 Hz, H-6), 6.23 (1H, d, J=2.4 Hz, H-
8)]. Moreover, there were also one aromatic signal at [δH 6.70 (1H, s,
H-6′)], a group of 2,2-dimethylpyran moiety at δH [5.66 (1H, d, J=9.8
Hz, H-3″′), 6.34 (1H, d, J=9.8 Hz, H-4″′), 1.44 (3H, s, H-5″′), 1.45 (3H,
s, H-6″′)] and a group of 2″′-hydroxy-3″′-methylbutenyl at [2.98 (1H,
dd, J=12.8, 8.5 Hz, Ha-1″), 2.88 (1H, overlapped, Hb-1″), 4.37 (1H, dd,
J=8.5, 4.3 Hz, H-2″), 4.91 (1H, brs, Ha-4″), 4.76 (1H, overlapped, Hb-
4″), 1.77 (3H, s, H-5″)]. In the HMBC spectrum, the key correlations of
H-1″/C-2′, C-3′, and H-4″′/C-4′, C-5′, C-6′ demonstrated the con-
nectivity of the 2″′-hydroxy-3″′-methylbutenyl and 2, 2-dimethylpyran
groups with C-2′, and C-4′, 5′, respectively (Fig. 2). Thus the planar
structure of 1 was established. In turn, compound 2, a stereoisomer of
1, was also obtained. It possessed the same molecular formula of
C25H28O5 and NMR spectroscopic data as 1, which suggested that the
only difference between them may be the absolute configuration. Their
stereochemistry of C-2 both involved a S-configuration from CD mea-
surements [16], while the configuration at C-2″ was difficult to confirm.
Consequently, it was only determined that 1 and 2 own opposite
daphnegiralin E and daphnegiralin F.
The HRESIMS spectra of 3 exhibited quasimolecular ion peaks at m/
z 433.1986 [M+Na]+ (calcd 433.1985) corresponding to the mole-
cular formula of C25H30O5. A comparison of the NMR spectroscopic
data of 3 with those of 1 and 2 revealed the existence of a 2, 2-di-
methyldihydropyran group at δH [1.84 (1H, t, J=6.8 Hz, H-3″), 2.85,
2.74 (each 1H, overlapped, H-4″), 1.35 (3H, s, H-5″), 1.37 (3H, s, H-6″)]
in ring B rather than a 2, 2-dimethylpyran moiety. The HMBC cross
peaks of H-4″/C-2′, C-3′, and H-1″′/C-4′, C-5′, C-6′ supported the idea
that the functional groups above were fused with the C-2′, 3′ and C-5′ of
ring B. Based on the Cotton effects in CD spectra, the absolute config-
uration of 3 was determined as 2S [16]. As a new compound, 3 were
given the trivial name daphnegiralin G.
Compounds 4 and 5 displayed the same molecular formula of
C25H30O4 as inferred from the [M+Na]+ and [M + H]+ ion peak (m/z
417.2036 in 4, calcd 417.2036; m/z 395.2217 in 5, calcd 395.2217).
Careful comparison of the NMR spectra involving the 1H, 13C, and 2D-
NMR of 4 and 5 displayed that they were a pair of epimers, having a
characteristic 7-hydroxylflavan skeleton with 2-(1-hydroxy-1-methy-
lethyl)dihydrofuran and prenyl groups in ring B. According to the
HMBC correlations of H-3″/C-3′, C-4′ and H-1″′/C-4′, C-5′, C-6′, the 2-
(1-hydroxy-1-methylethyl)dihydrofuran and prenyl substituents were
deduced to link at C-3′, 4′ and C-5′, respectively (Fig. 2). Analysis of the
CD data of 4 and 5 revealed that they had the same S absolute con-
figuration at C-2 [16]. However, several attempts to determine the
absolute configuration at C-2″ were unsuccessful. Therefore, these two
compounds were named daphnegiralin H and daphnegiralin I, respec-
tively.
Compound 6 was obtained as yellow solid. The molecular formula,
C25H28O5, was assigned by the quasi-molecular ion peak at m/z
431.1831 [M+Na]+ (calcd for C25H28O5Na, 431.1829) in HRESIMS.
The 1H and 13C spectroscopic data of 6 were similar to those of 4 and 5,
while having a 2, 2-dimethylpyran ring [δH 5.58 (1H, d, J=9.8 Hz, H-
3″′), 6.30 (1H, d, J=9.8 Hz, H-4″′), 1.46 (3H, s, H-5″′), 1.47 (3H, s, H-
6″′)] instead of a prenyl group. This group was fused at C-4′, 5′, as
indicated by HMBC cross peaks of H-4″′/C-4′, C-5′, C-6′, while the po-
sition of 2-(1-hydroxy-1-methylethyl)-3-hydroxy- dihydrofuran moiety
changed to C-2′, 3′ as deduced from the HMBC signals for H-3″/C-2′, C-
3′ (Fig. 2). The absolute configuration at C-2 was confirmed as S by
comparison of the CD values with literature data [16]. Hence, com-
pound 6 was named daphnegiralin K.
Compound 7 was obtained as yellow solid, and its molecular for-
mula was determined to be C25H30O6 by HRESIMS analysis, which gave
a m/z of 449.1936 for [M+Na]+ (calcd for C25H30O6Na, 449.1935).
The NMR data of 7 displayed the signal patterns characteristic of a 7-
hydroxyflavan with a prenyl [δH 3.32 (2H, m, H-1″), 5.04 (1H, t, J=6.9
Hz, H-2″), 1.61 (3H, s, H-4″), 1.65 (3H, s, H-5″)] connected to C-2′,
which was further supported by the HMBC long range correlations of H-
1″/ C-1′, C-2′, C-3′ and H-2/ C-1′, C-2′, C-6′ (Fig. 2). In addition, 7 had a
2-(1-hydroxy-1-methylethyl)-3- hydroxydihydrofuran moiety [δH 3.36

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Q. Sun et al. Fitoterapia 132 (2019) 68–74

Fig. 4. 6 induced apoptosis in Hep3B cells. (A) Cells were treated with different 6 concentrations for 48 h, and the apoptotic body of Hep3B cells was visualized
through Hoechst 33258 staining. (B) The percentage of apoptotic cells was analyzed through the Annexin V-PITC/PI assay after cells were treated with 6 for 48 h. (C)
Effects of 6 on Bcl-2, Bax and cl-PARP protein expression. Cells were treated with 6 for 48 h and analyzed with Western blot. The results are expressed as the
mean ± SD for each group. ⁎p < 0.05, ⁎⁎p < 0.01 for comparison with vehicle control.

(1H, overlapped, H-2″′), 4.29 (1H, t, J=6.0 Hz, H-3″′), 1.12 (3H, s, H-
5″′), 1.40 (3H, s, H-6″′)] and a hydroxyl group. In the HMBC spectra,
correlations of H-3″′/C-4′, C-5′, C-6′ indicated that the dihydrofuran
ring and hydroxyl group were located at the C-4′, 5′ and C-3′ in ring B,
respectively. The absolute configuration at C-2 of 7 was determined as S
based on its negative Cotton effect in the CD curve [16]. The relative
configuration at the C-2″′ and C-3″′ asymmetric carbon centers was
confirmed to be cis from the large vicinal proton-proton coupling con-
stant (J2″′, 3″′=6.0 Hz) [17]. Consequently, compound 7 was named
daphnegiralin L.
Compound 8 possessed the molecular formula C26H32O6, based on
the HRESIMS at m/z 463.2090 [M+Na]+ (calcd 463.2091). The 1H and
13
C NMR of 8 were similar to those of 7. However, the hydroxyl group
at C-3″′ in 7 was replaced by a methoxyl group, suggesting that 8 was a
3″′-OCH3 derivative of 7 through the HMBC cross peaks of OCH3-3″′/C-
3″′ (Fig. 2). The absolute 2S configuration of 8 was established by CD
spectroscopy [16]. The small coupling constant between H-2″′ and H-
3″′ (J2″′, 3″′=2.5 Hz) suggested that C-2″′ substituent and the C-3″′
methoxyl were trans-orientated [17]. On the basis of these observations,
compound 8 was given the name daphnegiralin M.
Compound 9 was isolated as a yellow solid with the molecular
formula C21H24O5 (HRESIMS, m/z 379.1510 [M+Na]+, calcd

73
Q. Sun et al.
379.1516). The 1H NMR spectra (Tables 1 and 3) displayed the pre-
sence of 2-(1-hydroxy-1-methylethyl)-3-hydroxydihydrofuran group at
δH [4.25 (1H, d, J=2.8 Hz, H-2″′), 4.98 (1H, d, J=2.8 Hz, H-3″′), 1.00
(3H, s, H-5″′), 1.19 (3H, s, H-6″′)], with the remaining signals being a
simple 7-hydroxyflavan. Resonances for ABX -coupling protons [δH
7.44 (1H, d, J=2.0 Hz, H-2′), 6.84 (1H, d, J=8.2 Hz, H-5′), 7.28 (1H,
dd, J=8.2, 2.0 Hz, H-6′)], as well as the HMBC correlations of H-2′/C-
3″ implied that the position of the dihydrofuran ring was C-3′, 4′ in ring
B, thus establishing the planar structure of 9 (Fig. 2). Analysis of the CD
data and coupling constant (J2″′, 3″′=2.8 Hz) revealed that 9 had S
absolute configuration at C-2 and trans relative configuration at the C-
2″′ substituent and C-3″′ methoxyl [16,17]. Compound 9 was then
named daphnegiralin N.
In our study, we evaluated the cytotoxic potency of flavan deriva-
tives against six human cancer cell lines using MTT assay. As shown in
Table 4, 6 had most potent cytotoxic effect against Hep3B cells with an
IC50 of 9.83 μM. Moreover, 6 inhibited the growth of Hep3B cells in a
concentration-dependent manner, it had comparable anti-tumor ac-
tivity on Hep3B cells with the positive group (Fig. 3).
Apoptosis is a key form of cell death induced by chemotherapeutic
agents [18]. The above result indicated compound 6 had potent effect
against Hep3B cells. Therefore, the apoptosis mechanism of 6-treated
Hep3B cells was investigated in the subsequent experiment (Fig. 4).
Hoechst 33258 staining was performed to elucidate the cytotoxicity of
6. Compared with the control group, 6-treated cells at 5, 10, 20 μM
exhibited nuclear condensation and fragmentation which were the
hallmarks of apoptotic cells. The results determined that 6 might induce
the apoptosis of Hep3B cells.
Thereafter, Annexin V-FITC/PI double staining was applied to
measure the ratio of apoptotic cells in 6-treated Hep3B cells using flow
cytometer analysis. After 48 h of 6 treatment, the compound at the dose
of 5, 10, 20 μM caused 14.85%, 61.63%, 88.76% apoptosis in Hep3B
cells respectively. Notably, we found the compound 6 had remarkable
effects on inducing apoptosis in Hep3B cells.
To further study the mechanism of 6-induced apoptosis, western
blot analysis was performed in Hep3B cells. These results implied that 6
caused up-regulations of Bax and cl-PARP, whereas decreased Bcl-2
expression. Consequently, our results demonstrated that 6 markedly
induced apoptosis in Hep3B cells.
In summary, nine new compounds were isolated from stem and root
bark of Daphne giraldii. Their structures were determined by extensive
spectroscopic analyses. Among the isolates, 6 displayed significant cy-
totoxic activity against Hep3B cells comparable with the positive con-
trol 5-Fu. By means of Hoechst 33258 staining, Annexin V-FITC/PI
double staining and Western blot analysis, we discovered that
74
Fitoterapia 132 (2019) 68–74
compound 6 had remarkable effects on inducing apoptosis in Hep3B
cells which may related to up-regulations of Bax and cl-PARP and a
decrease in Bcl-2 expression. These investigations indicated that flavans
with multi-chiral centers from D. giraldii are natural ingredients with
great potential for hepatocellular carcinoma therapy.
Acknowledgements
This research work was supported by National Nature Science
Foundation of China (81573319, 81673324, 81872766), China
Postdoctoral Science Foundation (2017M620104). Professor Y. Peng,
Mrs. W. Li and Mr. Y. Sha of Shenyang Pharmaceutical University are
appreciated for their help in measuring the HRESIMS and NMR data.
Appendix A. Supplementary data
Supplementary data to this article can be found online at https://
doi.org/10.1016/j.fitote.2018.11.011.
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