Phytochemistry 172 (2020) 112255

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Phytochemistry
journal homepage: www.elsevier.com/locate/phytochem

Cytotoxic sesquiterpene glucosides from Fissistigma pallens T

c,d c,e c c a
Ngo Sy Thinh , Nguyen Thi Bich Thu , Tran Minh Ngoc , Nguyen Minh Khoi , Bui Huu Tai ,
Phan Van Kiema, Chau Van Minha, Nguyen Xuan Nhiema,∗∗, Yohan Seob, Wan Namkungb,
SeonJu Parkb, Seung Hyun Kimb,∗
a
Institute of Marine Biochemistry (IMBC), Vietnam Academy of Science and Technology (VAST), 18 Hoang Quoc Viet, Cau Giay, Hanoi, Viet Nam
b
Yonsei Institute of Pharmaceutical Sciences, College of Pharmacy, Yonsei University, Incheon, 406-840, South Korea
c
National Institute of Medicinal Materials, 3B Quang Trung, Hoan Kiem, Hanoi, Viet Nam
d
354 Military Hospital, 120 Doc Ngu, Ba Dinh, Hanoi, Viet Nam
e
College of Pharmacy, Duy Tan University, 254 Nguyen Van Linh, Thanh Khe, Da Nang, Viet Nam

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

Keywords: Six undescribed sesquiterpene glucosides, fissispallins A-F, and one known sesquiterpene glucoside, fissispallin,
Fissistigma pallens were discovered in the leaves of Fissistigma pallens (Finet & Gagnep.) Merr. The structures were determined using
Annonaceae spectroscopic methods, including 1D, 2D NMR, and MS. All compounds were evaluated for cytotoxic activity
Sesquiterpene against three human cancer cell lines, HT-29, A-2058, and A-549. Fissispallin A showed potent activity with the
Fissispallin
IC50 values less than 1.5 μM against all tested human cancer cell lines. Fissispallin also showed potent activity
Cytotoxic activity
with IC50 value of 0.4 ± 0.3 on the A-2058 cancer cell lines. Fissispallins B-D showed significant cytotoxic
activity against all the tested cancer cell lines with IC50 values ranging from 3.8 to 7.2 μM.

1. Introduction
Fissistigma is a genus of the plant family Annonaceae that includes
66 species of which 23 species of Fissistigma are distributed in Vietnam
(Ban, 2000). Some species have been found to contain alkaloids (Chia
et al., 1998; Wu et al., 1990) and sesquiterpenes (Porzel et al., 2000). In
addition, the compounds and extracts from the Fissistigma species have
been found to exhibit cytotoxic (Fan et al., 2012) and anti-in-
flammatory effects (Ge et al., 2013). The essential oil extracted from
some species of this genus is also used as a fragrance. Essential oils of
this species are primarily comprised of sesquiterpenes (Thang et al.,
2014).
Fissistigma pallens (Finet & Gagnep.) Merr. (Annonaceae) is an im-
portant climbing shrub that grows in the Northern regions of Vietnam.
Other plants in the Fissistigma genus, including F. pallens provide an
essential oil fragrance. F. pallens has been used in traditional medicine
to treat diseases like muscular atrophy, hepatomegaly, hepatospleno-
megaly, traumatic injuries, sciatic muscle strains, arthritis, rheumatism,
and asthma. Recent phytochemical research has shown that F. pallens
contains alkaloids (Höferl et al., 2013) that contribute to anti-in-
flammatory and anti-cancer activities. Therefore, it is necessary to un-
derstand the constituents and function of organic compounds contained
in the plant. Here, we report the structure of six undescribed sesqui-
terpene glucosides and one known compound. We also report and their
cytotoxic activities.
2. Results and discussion
The leaves of F. pallens were sonicated with hot methanol to yield
methanol (MeOH) extract. The MeOH extract was suspended in H2O
and successively partitioned with n-hexane, chloroform, and ethyl
acetate (EtOAc) to yield the layers. Six undescribed sesquiterpene glu-
cosides and one known compound were isolated using various chro-
matographic resin and isolation techniques.
Compound 1 was obtained as a white amorphous powder and its
molecular formula was determined to be C30H42O7 from HRESIMS at
m/z 537.2822 [M+Na]+ (Calcd. for [C30H42O7Na]+, 537.2823). The
1
H NMR spectrum of 1 (in CDCl3) showed the proton signals of a vinyl
group at δH 4.78 (d, J = 17.2 Hz), 4.80 (d, J = 11.2 Hz), and 5.72 (dd,
J = 11.2, 17.2 Hz), two methylene olefin protons at δH 4.47 (s) and
4.69 (s), four methyl groups at δH 0.87, 1.12, 1.17, and 1.59 (each 3H,
s); two olefinic protons; five aromatic protons; and one anomeric
proton. The 13C NMR and DEPT spectra of 1 displayed the signals of 30
carbons, including one carbonyl, four non-protonated carbons, fifteen

∗
Corresponding author. College of Pharmacy, Yonsei Institute of Pharmaceutical Sciences, Yonsei University, Incheon, 21983, South Korea.
∗∗
Corresponding author. Institute of Marine Biochemistry, Vietnam Academy of Science and Technology (VAST), 18 Hoang Quoc Viet, Cau Giay, Hanoi, Viet Nam.
E-mail addresses: nxnhiem@yahoo.com (N.X. Nhiem), kimsh11@yonsei.ac.kr (S.H. Kim).

https://doi.org/10.1016/j.phytochem.2019.112255
Received 4 May 2019; Received in revised form 29 December 2019; Accepted 30 December 2019
0031-9422/ © 2020 Elsevier Ltd. All rights reserved.
N.S. Thinh, et al.
methines, six methylenes, and four methyl carbons, suggesting the
presence of one sesquiterpene, one cinnamoyl, and one monosaccharide
moiety. Analysis of the 1H and 13C NMR spectra of 1 indicated that its
structure was similar to α-elemol 11-O-β-D-fucopyranoside (D'Ambrosio
et al., 2015) except for the acylglucopyranosyl moiety at C-11. The
HMBC correlations (Fig. 2) between H-14 (δH 0.87) and C-1 (δC 150.2)/
C-5 (δC 52.5)/C-10 (δC 39.7); H-15 (δH 1.59) and C-3 (δC 112.0)/C-4 (δC
147.7)/C-5 (δC 52.5); H-12 (δH 1.12)/H-13 (δH 1.17) and C-7 (δC 48.1)/
C-11 (δC 80.4) suggested the presence of cyclohexane linked with iso-
propenyl and isopropyl groups at C-5 and C-7, respectively, and both
vinyl and methyl groups at C-10. The coupling constants of H-5 and Hα-
6/Hβ-6, J = 3.2 and 12.8 Hz suggested the configuration of H-5 is an
axial orientation (α). In addition, the configurations of H-5, H-7, and the
methyl group at C-10 were determined to be axial orientation by
NOESY correlations between H-5 (δH 1.86) and H-2 (δH 4.78)/H-7 (δH
1.38). The monosaccharide was shown to be D-glucose using acid hy-
drolysis (identified as trimethylsilyl derivative by GC method) (Thu
et al., 2015). The large coupling constant between glc H-1′ and glc H-2′,
J = 8.0 Hz confirmed the configuration of anomeric proton as axial
orientation (β-D-glucopyranosyl). In addition, the position glucose
moiety at C-11 was confirmed by HMBC correlations from glc H-1′ (δH
4.68) to C-11 (δC 80.4). The HMBC correlations between H-2′ (δH 4.88)
and C-9′′ (δC 166.6) suggested the presence of cinnamoyl at C-2 of
glucopyranosyl. Based on the above evidence, the constitution of 1 was
established to be elemol 11-O-(2-(E)-cinnamoyl)-β-D-glucopyranoside
and named fissispallin A.
The molecular formula of compound 2 was determined as C30H44O8
based on the HRESIMS sodium adduct ion at m/z: 555.2934 [M+Na]+
(Calcd. for [C30H44O8Na]+, 555.2928). The 1H NMR spectrum of 2
exhibited proton signals of four methyl groups at δH 0.76, 1.03, 1.06,
and 1.16 (each 3H, s), suggesting the presence of a sesquiterpene
aglycone, one cinnamoyl moiety, and one anomeric proton at δH 4.65
(1H, d, J = 7.8 Hz). The 13C NMR spectrum of 2 showed the signals of
30 carbons. Of these, six carbon signals were assigned to glucopyr-
anosyl, nine to cinnamoyl, and fifteen to sesquiterpene aglycone.
Analysis of the 1H and 13C-NMR data of 2 indicated that the aglycone
was identical to proximadiol (Evans et al., 1982; Harinantenaina et al.,
2006). The locations and configurations of functional groups were
elucidated by extensive 2D NMR analysis. The HMBC correlations
(Fig. 2) between H-15 (δH 1.06) and C-3 (δC 42.3)/C-4 (δC 72.9)/C-5 (δC
53.8) suggested the position of the hydroxyl group at C-4. The HMBC
correlations between H-12 (δH 1.03)/H-13 (δH 1.16) and C-7 (δC 48.1)/
C-11 (δC 81.1) suggested the presence of an isopropyl group at C-7.
Furthermore, the β-D-glucopyranosyl position at C-11 was confirmed by
HMBC correlations from glc H-1′ (δH 4.65) to C-11 (δC 81.1). The po-
sition of the cinnamoyl group at C-2′ of glucopyranosyl was confirmed
by HMBC correlation from H-2′ (δH 4.91) to C-9′′ (δC 166.0). The axial
orientation of the methyl groups at C-4 and C-10, H-5, and H-7 were
shown by the NOESY observations between H-14 (δH 0.76) and H-15
(δH 1.06); H-5 (δH 1.18) and H-7 (δH 1.51) (Fig. 3). The sesquiterpene
aglycone skeleton of 2 was identified as eudesmane-4α,11-diol by
comparing its 13C NMR at C-7 (δC 48.2) to ent-eudesmane sesqui-
terpene-type, prerodontoside A [δC 42.5 (C-7)] (Li et al., 1998) and
eudesmane sesquiterpene-type, celerodie E [δC 48.2 (C-7)] (Kitajima
et al., 2003). Therefore, the structure of 2 was determined as 4α-hy-
droxyeudesmane 11-O-(2-(E)-cinnamoyl)-β-D-glucopyranoside and
named fissispallin B.
The molecular formula of compound 3 was identified as C31H46O8
by HRESIMS sodium adduct ion at m/z: 569.3085 [M+Na]+ (Calcd. for
[C31H46O8Na]+, 569.3084). The 1H NMR spectrum of 3 indicated a
sesquiterpene aglycone with four methyl groups at δH 0.82, 1.00, 1.05,
and 1.16 (each 3H, s), one (E)-cinnamoyl moiety including two olefinic
protons, five aromatic protons; and one anomeric proton at δH 4.65
(1H, d, J = 8.0 Hz); and one methoxy group at δH 3.16 (s). Comparison
of the 1H and 13C NMR data to 2 indicated the structure of 3 was similar
to 2 with the addition of methoxy group at C-4. The position of methoxy
2
Phytochemistry 172 (2020) 112255
group at C-4 was shown by the HMBC correlations between H-15 (δH
1.00) and C-3 (δC 37.5)/C-4 (δC 76.9)/C-5 (δC 52.8) and between
methoxy (δH 3.16) and C-4 (δC 76.9). The axial orientation of H-5, H-7,
and the methyl groups at C-4 and C-10 were confirmed by the NOESY
observations between H-14 (δH 0.82) and Hβ-2 (δH 1.38)/H-15 (δH
1.00); H-5 (δH 1.17) and H-7 (δH 1.52) (Fig. 3). Therefore, the structure
of 3 was determined as 4α-methoxyeudesmane 11-O-(2-(E)-cinnamoyl)-
β-D-glucopyranoside and named fissispallin C.
The HRESIMS of 4 showed a sodium adduct ion peak at m/z
537.2824 (Calcd. for [C30H42O7Na]+, 537.2823), resulting in the mo-
lecular formula of C30H42O7. Analysis of 1D and 2D NMR spectra of 4
determined it comprised the moieties of one sesquiterpene, one cinna-
moyl, and one monosaccharide. The characteristic signals in the 1H and
13
C NMR spectra and acid hydrolysis of 4 identified the sugar moiety as
β-D-glucopyranose. Furthermore, the structure of 4 was found to be
similar to 2′-O-acetyl-4-O-β-D-glucopyranosyl–germacra-1 (10),5-diene,
except for the acetyl moiety replaced by a cinnamoyl moiety. This was
further confirmed by the HMBC correlations from glc H-2′ to cin C-9′′.
The HMBC correlations from H-12/H-13 to C-7/C-11, from H-14 to C-1/
C-9/C-10, and from H-15 to C-3/C-4/C-5 indicated the positions of the
methyl group at C-4, the isopropyl group at C-11, and two double bonds
at C-5/C-6 and C-1/C-10. The configurations of both double bonds at C-
1/C-10 and C-5/C-6 were shown as E by the NOESY observations be-
tween H-1 (δH 4.74) and H-9 (δH 2.12) and the large coupling constant
of H-5 and H-6, J = 16.0 Hz and (Fig. 3). Moreover, the β-orientation of
methyl and isopropyl groups at C-4 and C-7 were confirmed by NOESY
correlations of Hα-3 (δH 1.67)/H-5 (δH 4.97)/H-7 (δH 1.90), H-6 (δH
5.10)/H-15 (δH 1.31), H-14 (δH 1.41) and H-15 (δH 1.31). Based on this
evidence, the structure of 4 was defined as germacra-1(10),5-diene-4-O-
(2-(E)-cinnamoyl)-β-D-glucopyranoside and named fissispallin D.
The 1H and 13C NMR spectra of 5 exhibited the presence of one
sesquiterpene aglycone, one sugar moiety, and one cinnamoyl moiety.
A literature search revealed that the structure of 5 was similar to un-
dulatumoside B (Mendes et al., 2013) with a different substitution
group at C-11. The four methyl groups, including two tertiary methyl
groups at δH 1.15 (s) and 1.18 (s) and two secondary methyl groups at
δH 0.90 (d, J = 7.2 Hz) and 0.93 (d, J = 7.2 Hz) were identified in the
1
H NMR spectrum. The HMBC correlations from H-15 (δH 0.90) to C-3
(δC 30.8)/C-4 (δC 46.2)/C-5 (δC 139.0), H-6 (δH 1.84 and 2.15) to C-1
(δC 139.7)/C-4 (δC 46.2)/C-5 (δC 139.0), and from H-14 (δH 0.93) to C-1
(δC 139.7)/C-9 (δC 33.7)/C-10 (δC 33.6) showed the fused bicyclic rings
of cyclopentane and cycloheptane at C-1/C-5, identified as the guaiane-
type sesquiterpene, guaian-1(5)-en-11-ol (Mendes et al., 2013;
Raharivelomanana et al., 1995). The relative stereochemistry of ses-
quiterpene aglycone 5 was deduced from the similar 1H and 13C NMR
data of undulatumoside B (Mendes et al., 2013) and NOESY observa-
tions. The observation of NOESY interactions (Fig. 3) between H-4 (δH
2.50) and Hα-3 (δH 1.91)/Hα-6 (δH 2.15), H-7 (δH 1.72) and Hα-6 (δH
2.15), and between Hβ-8 (δH 1.38) and Hβ-6 (δH 1.84)/H-14 (δH 0.93)
indicated that the orientations of H-4, H-7, and H-10 were on the same
side of molecule. Like 1–3, the acylglycopyranoside of 5 was de-
termined as 6-(E)-cinnamoyl)-β-D-glucopyranoside attached to C-11 of
guai-1(10)-ene by the HMBC correlations from glc H-6′ (δH 4.40) to cin
C-9′′ (δC 167.3) and from glc H-1′ (δH 4.45) to C-11 (δC 81.3). Thus, the
structure of fissispallin E (5) was elucidated as guai-1(5)-en-11-O-(6-
(E)-cinnamoyl-β-D-glucopyranoside.
The HRESIMS experiment of 6 resulted in the same molecular for-
mula as 5. Compound 5 contained a small amount of 6 (20%). The 1H
and 13C NMR data of 6 were nearly identical to 5, the only difference
was a shift of the double bond position from C-1/C-5 to C-1/C-10. The
sesquiterpene was found to be bulnesol (Raharivelomanana et al.,
1995). The positions of methyl groups at C-4, C-10, and C-11 were also
supported by HMBC correlations. In addition, acylglucoside was de-
termined to be 6-(E)-cinnamoyl)-β-D-glucopyranoside at the C-11 of
bulnesol. The NOESY correlations between H-5 (δH 2.36) and Hα-3 (δH
1.58)/H-4 (δH 2.04)/H-7 (δH 1.60) and between Hβ-6 (δH 0.69) and H-
N.S. Thinh, et al.
Fig. 1. Chemical structures of compounds 1–7.
15 (δH 0.85)/H-13 (δH 1.12) suggested that the protons were oriented
on the same side (α-configurations). Thus, the structure of fissispallin F
(6) was determined to be bulnesol-11-O-(6-(E)-cinnamoyl)-β-D-gluco-
pyranoside.
The known compound was characterized as fissispallin (7) (Thuy
et al., 2006) by comparing its observed and reported physical data
(Fig. 1).
The cytotoxic activity of compounds 1–7 were evaluated against
three human cancer cell lines: HT-29 (colon cancer), A-2058 (skin
melanoma cancer), A-549 (lung cancer) (Table 3). Compounds 1–4 and
7 showed significant cytotoxic activity in all human cancer cell lines
with IC50 values ranging from 0.4 to 7.8 μM, compared to the positive
control, mitoxantrone (IC50 values ranging from 7.8 to 10.3 μM).
Compounds 5 and 6 were guaiane-type compounds that exhibited
moderate cytotoxic activity against three cancer cell lines with IC50
values ranging from 15.5 to 23.9 μM. Among the active compounds,
compound 4, a germacrene derivative, showed similar activity to that
of eudesmane derivatives, compounds 2–3, and 7. Compound 1 showed
the most potent activity with IC50 values less than 1.5 μM against all
tested cancer cell lines. Compound 1 contains an (−)-elemol structure,
which is a hydroxylated derivative of (−)-trans-β-elemene. Recent re-
ports have shown the significant cytotoxic effect of (−)-trans-β-elemene
even against drug-resistant tumors with fewer side effects (Adio, 2009).
β-Elemene was approved as a non-cytotoxic class II antitumor drug, and
it has primarily been used as an adjunctive drug to enhance the efficacy
and reduce the toxicity of chemoradiotherapy, and to reverse drug re-
sistance (Zhai et al., 2018). These results suggest that the discovery of
unprecedented (−)-trans-β-elemene and related compounds may in-
crease the possibility of finding anticancer agents. The mechanism of
action of compound 1 should be studied further.
3. Experimental
3.1. General
All NMR spectra were recorded on an AGILENT 400 MHz or
3
Phytochemistry 172 (2020) 112255
800 MHz. Data processing was performed with MestReNova ver. 9.0.1.
HRESIMS spectra were obtained using an AGILENT 6550 iFunnel Q-
TOF LC/MS system. HPLC was performed using an AGILENT 1200
HPLC system. Column chromatography (CC) was performed on silica-
gel (Kieselgel 60, 230–400 mesh, Merck) or RP-18 resins (30–50 μm,
Fuji Silysia Chemical Ltd.). For thin layer chromatography (TLC), pre-
coated silica-gel 60 F254 (0.25 mm, Merck) and RP-18 F254S (0.25 mm,
Merck) plates were used.
3.2. Plant material
The leaves of Fissistigma pallens (Finet & Gagnep.) Merr. were col-
lected at Que Phong, Nghe An, Vietnam in April 2016, and identified by
Dr. Nguyen The Cuong, Institute of Ecology and Biological Resources,
VAST. A voucher specimen (NCCT-P14) was deposited at the Institute
of Marine Biochemistry, VAST.
3.3. Extraction and isolation
The dried powdered leaves of F. pallens (5.0 kg) were sonicated
twice with hot methanol (MeOH) (each 10 L, 4 h, 50 °C) and the solvent
was removed in vacuo to yield MeOH extract (400.0 g). The MeOH
extract was suspended in H2O (2.5 L) and successively partitioned with
n-hexane, chloroform, and ethyl acetate (EtOAc) to yield n-hexane
(FP1, 14.0 g), chloroform (FP2, 72.0 g), EtOAc (FP3, 9.0 g) residues,
and a water layer (FP4 g).
The FP2 fraction was loaded on a silica gel column and eluted with a
solvent system of n-hexane‒acetone (100:1, 40:1, 20:1, 10:1, 5:1, 2.5:1,
1:1, v/v) to give seven smaller fractions: FP2A (3.0 g), FP2B (7.0 g),
FP2C (4.5 g), FP2D (4.1 g), FP2E (5.0 g), and FP2F (7.5 g), respectively.
The FP2F fraction was chromatographed on a silica gel CC, eluting with
chloroform‒MeOH (10:1, v/v) to give four sub-fractions, FP2F1 (0.8 g),
FP2F2 (3.5 g), FP2F3 (0.7 g), and FP2F4 (0.6 g). The FP2F2 fraction
was chromatographed on a RP-18 CC, using chloroform‒MeOH (10:1,
v/v) as the eluent solvent to give three smaller fractions: FP2F2 A
(0.4 g), FP2F2B (0.2 g), and FP2F2C (0.6 g).
N.S. Thinh, et al. Phytochemistry 172 (2020) 112255

Fig. 2. The key HMBC and COSY correlations of compounds 1–6.

The FP2F2 A fraction was chromatographed on an HPLC column from FP2F2B fraction using an HPLC column (J'sphere, ODS H-80,
(J'sphere, ODS H-80, 4 μM, 250 × 20 mm) with a flow rate of 4 mL/min 4 μM, 250 × 20 mm) with flow rate of 4 mL/min eluting 80% acet-
eluting 80% acetonitrile to yield compounds 2 (8.0 mg) and 3 onitrile. The purification of the FP2F2C fraction using an HPLC column
(13.0 mg). Compounds 1 (16.0 mg), and 4 (33.0 mg) were obtained (J'sphere, ODS H-80, 4 μM, 250 × 20 mm) with flow rate of 4 mL/min

Fig. 3. The key NOESY correlations of compounds 1–6.

4
N.S. Thinh, et al. Phytochemistry 172 (2020) 112255

Table 1
1
H and13C NMR spectroscopic data for compounds 1–3 in CDCl3.
C 1 2 3

δC a
δH (mult., J = Hz)
b
δC a
δH (mult., J = Hz)
b
δC a
δHb (mult., J = Hz)

Aglycone
1 150.2 5.72 (dd, 11.2, 17.2) 40.8 1.04 (ddd, 4.0, 12.8, 12.8, α) 40.6 1.03 (ddd, 4.0, 12.8, 12.8, α)
1.29 (dt, 4.0, 4.0, 12.8, β) 1.31 (ddd, 2.4, 4.0, 12.8, β)
2 110.0 4.78 (d, 17.2) 20.0 1.39 (m, β) 19.5 1.38 (m, β)
4.80 (d, 11.2) 1.49 (m, α) 1.52 (m, α)
3 112.0 4.47 (s)/4.69 (s) 42.3 1.38 (m, β) 37.5 1.19 (d, 4.0, 12.8, 13.6, β)
1.70 (br d, 12.8, α) 1.91 (dt, 4.0, 4.0, 12.8, α)
4 147.7 – 72.9 – 76.9 –
5 52.5 1.86 (dd, 3.2, 12.8) 53.8 1.18 (dd, 2.4, 12.0) 52.8 1.17 (m)
6 27.9 1.29 (m, β) 20.8 0.86 (dd, 12.0, 12.4, β) 20.8 0.88 (ddd, 8.8, 12.0, 12.8, β)
1.56 (m, α) 2.13 (br d, 12.4, α) 2.20 (br d, 12.8, α)
7 48.1 1.38 (m) 48.1 1.51 (m) 47.1 1.52 (m)
8 22.5 1.54 (m) 23.7 1.11 (ddd, 4.0, 12.8, 12.8, β) 23.5 1.13 (m, β)
1.37 (m, α) 1.38 (m, α)
9 39.9 1.32 (m) 44.7 1.14 (m, α) 45.0 1.17 (m, α)
1.34 (m, β) 1.35 (dt, 3.2, 12.0, β)
10 39.7 – 34.5 – 34.4 –
11 80.4 – 81.1 – 81.1 –
12 22.6 1.12 (s) 22.0 1.03 (s) 22.5 1.05 (s)
13 25.1 1.17 (s) 25.4 1.16 (s) 25.5 1.16 (s)
14 16.6 0.87 (s) 18.7 0.76 (s) 19.2 0.82 (s)
15 24.6 1.59 (s) 22.9 1.06 (s) 16.3 1.00 (s)
4-OMe 47.8 3.16 (s)
11-OGlc
1′ 95.2 4.68 (d, 8.0) 95.0 4.65 (d, 7.8) 94.8 4.65 (d, 8.0)
2′ 74.2 4.88 (t, 8.0) 74.5 4.91 (dd, 7.8, 9.3) 74.7 4.89 (dd, 8.0, 8.8)
3′ 75.6 3.68 (dd, 8.0, 8.8) 75.6 3.68 (dd, 8.8, 9.3) 76.2 3.63 (t, 8.8)
4′ 70.8 3.66 (dd, 8.0, 8.8) 70.7 3.60 (dd, 8.8, 8.8) 71.7 3.45 (dd, 7.2, 8.8)
5′ 75.1 3.35 (m) 76.4 3.37 (m) 76.5 3.38 (m)
6′ 62.2 3.83 (m) 61.7 3.78 (dd, 5.6, 12.4) 62.6 3.66 (dd, 7.2, 12.0)
3.89 (dd, 2.4, 12.4) 3.91 (br d, 12.0)
Cin
1″ 134.2 – 134.2 – 134.2 –
2″, 6″ 128.2 7.45 (d, 8.0) 128.2 7.48 (d, 8.0) 128.2 7.49 (d, 8.0)
3″, 5″ 128.8 7.33 (t, 8.0) 128.9 7.35 (d, 8.0) 128.9 7.37 (t, 8.0)
4″ 130.4 7.34 (t, 8.0) 130.4 7.36 (t, 8.0) 130.5 7.38 (t, 8.0)
7″ 145.9 7.66 (d, 16.0) 146.0 7.71 (d, 16.0) 146.1 7.72 (d, 16.0)
8″ 117.4 6.39 (d, 16.0) 117.5 6.44 (d, 16.0) 117.3 6.43 (d, 16.0)
9″ 166.6 – 166.0 – 166.8 –

a
Recorded in 100 MHz;b)recorded in 800 MHz; assignments were done by HSQC, HMBC, COSY, and NOESY experiments; Glc, glucopyranosyl; Cin, cinnamoyl.

eluting 80% acetonitrile in water yielded compounds 5 (29.0 mg), 6 3.3.5. Fissispallin E (5)
(23.0 mg), and 7 (10.0 mg). White amorphous powder. Molecular formula: C30H42O7. HRESIMS
m/z: 537.2814 [M+Na]+ (Calcd. for [C30H42O7Na]+, 537.2823). 1H
3.3.1. Fissispallin A (1) and 13C NMR (CDCl3): see Table 2.
White amorphous powder. [α ]25
D = −300.0 (c 1.0 MeOH).
Molecular formula: C30H42O7. HRESIMS m/z: 537.2822 [M+Na]+ 3.3.6. Fissispallin F (6)
(Calcd. for [C30H42O7Na]+, 537.2823). 1H and 13C NMR (CDCl3): see White amorphous powder. [α ]25
D = +70.0 (c 1.0 MeOH). Molecular
Table 1. formula: C30H42O7. HRESIMS m/z: 537.2814 [M+Na]+ (Calcd. for
[C30H42O7Na]+, 537.2823). 1H and 13C NMR (CDCl3): see Table 2.
3.3.2. Fissispallin B (2)
White amorphous powder. [α ]25
D = −170.0 (c 1.0 MeOH). 3.4. Acid hydrolysis
Molecular formula: C30H44O8. HRESIMS m/z: 555.2934 [M+Na]+
(Calcd. for [C30H44O8Na]+, 555.2928). 1H and 13C NMR (CDCl3): see Each compound (1–6, 2.0 mg) was separately dissolved in 1.0 N HCl
Table 1. (dioxane−H2O, 1:1, v/v, 1.0 mL) and heated to 80 °C in a water bath
for 3 h. The acidic solution was neutralized with silver carbonate, and
3.3.3. Fissispallin C (3) the solvent thoroughly removed overnight under a nitrogen stream.
White amorphous powder. [α ]25
D = -240.0 (c 1.0 MeOH). Molecular After extracting with CHCl3, the water layer was concentrated to dry-
formula: C31H46O8. HRESIMS m/z: 569.3085 [M+Na]+ (Calcd. for ness using N2. The residue was dissolved in pyridine (0.1 mL) and then
[C31H46O8Na]+, 569.3084). 1H and 13C NMR (CDCl3): see Table 1. adding of L-cysteine methyl ester hydrochloride in pyridine (0.06 M,
0.1 mL). The reaction mixture was heated at 60 °C for 2 h and then
3.3.4. Fissispallin D (4) adding trimethylsilylimidazole solution (0.1 mL), and heating at 60 °C
White amorphous powder. [α ]25
D = +340.0 (c 1.0 MeOH). for 1.5 h. The dried product was partitioned with n-hexane and H2O
Molecular formula: C30H42O7. HRESIMS m/z: 537.2824 [M+Na]+ (0.1 mL each), and the organic layer was analyzed using GC with the
(Calcd. for [C30H42O7Na]+, 537.2823). 1H (800 MHz) and 13C NMR following conditions: column DB-5 (0.32 mm ID × 30 m length), de-
(100 MHz) (CDCl3): see Table 2. tector FID, column temp 210 °C, injector temp 270 °C, detector temp

5
N.S. Thinh, et al. Phytochemistry 172 (2020) 112255

Table 2
1
H and13C NMR spectroscopic data for compounds 4–6 in CDCl3.
C 4 5 6

δC a
δH (mult., J = Hz)
b
δC a
δH (mult., J = Hz)
b
δC a
δHb (mult., J = Hz)

Aglycone
1 129.2 4.74 (br d, 11.8) 139.7 – 141.9 –
2 24.0 1.92 (m, α) 35.4 2.05 (m, α) 30.4 2.09 (m, α)
2.17 (m, β) 2.38 (m, β) 2.25 (m, β)
3 34.3 1.48 (dt, 4.0, 13.4, β) 30.8 1.22 (m, β) 33.0 1.28 (m, β)
1.67 (dt, 3.8, 13.4, α) 1.91 (m, α) 1.58 (m, α)
4 81.4 – 46.2 2.50 (m) 38.9 2.04 (m)
5 137.9 4.97 (d, 16.0) 139.0 – 46.1 2.36 (m)
6 134.1 5.10 (dd, 9.8, 16.0) 27.7 1.84 (dd, 13.5, 13.5, β) 28.5 0.69 (dd, 11.6, 12.0, β)
2.15 (br d, 13.5, α) 1.91 (br d, 12.0, α)
7 53.6 1.90 (m) 47.4 1.72 (m) 52.2 1.60 (m)
8 26.6 1.37 (m) 27.1 1.38 (m, β)/1.76 (m, α) 28.0 0.98 (m, α)/1.80 (m, β)
9 41.2 2.12 (m) 33.7 1.65(m, β)/1.51(m, α) 35.1 2.00 (m, α)/2.09 (m, β)
10 131.8 – 33.6 2.24 (m) 128.4 –
11 32.7 1.44 (m) 81.3 – 81.6 –
12 19.3 0.81 (d, 6.8) 24.9 1.15 (s) 24.6 1.19 (s)
13 21.2 0.88 (d, 6.6) 24.4 1.18 (s) 23.6 1.12 (s)
14 16.6 1.41 (s) 19.7 0.93 (d, 7.2) 22.3 1.57 (s)
15 20.8 1.31 (s) 20.0 0.90 (d, 7.2) 15.4 0.85 (d, 6.8)
11-OGlc
1′ 95.6 4.56 (d, 8.0) 96.9 4.45 (d, 8.0) 96.9 4.47 (d, 8.0)
2′ 74.0 4.85 (dd, 8.0, 9.6) 73.7 3.37 (dd, 8.0, 8.8) 73.7 3.38 (dd, 8.0, 8.8)
3′ 75.5 3.56 (t, 9.6) 76.4 3.58 (dd, 8.8, 9.2) 76.4 3.60 (dd, 8.0, 8.8)
4′ 70.3 3.65 (t, 9.6) 70.4 3.42 (dd, 8.8, 9.2) 70.4 3.42 (dd, 8.0, 8.8)
5′ 75.4 3.24 (dt, 3.6, 9.6) 73.7 3.47 (m) 73.8 3.52 (m)
6′ 61.0 3.82 (m) 63.9 4.40 (m) 64.1 4.40 (dd, 6.0, 12.0)
4.46 (br d, 12.0)
Cin
1″ 134.3 – 134.3 – 134.2 –
2″, 6″ 128.1 7.52 (d, 8.0) 128.1 7.48 (d, 8.0) 128.1 7.47 (d, 8.0)
3″, 5″ 128.9 7.38 (t, 8.0) 128.8 7.34 (t, 8.0) 128.8 7.35 (t, 8.0)
4″ 130.4 7.38 (t, 8.0) 130.3 7.34 (t, 8.0) 130.3 7.35 (t, 8.0)
7″ 145.6 7.71 (d, 16.0) 145.4 7.66 (d, 16.0) 145.5 7.66 (d, 16.0)
8″ 117.7 6.44 (d, 16.0) 117.6 6.41 (d, 16.0) 117.5 6.41 (d, 16.0)
9″ 166.4 – 167.3 – 167.3 –

a
Recorded in 100 MHz.
b
Recorded in 800 MHz; assignments were done by HSQC, HMBC, COSY, and NOESY experiments; Glc, glucopyranosyl; Cin, cinnamoyl.

Table 3 Three human cancer cell lines, including HT-29 (colon cancer), A-2058
Cytotoxic effects of compounds 1–7. (skin melanoma cancer), and A-549 (lung cancer) were grown in RPMI
Compounds IC50a
1640 medium supplemented with 10% fetal bovine serum and peni-
cillin/streptomycin (100 U/mL and 100 g/mL, respectively) at 37 °C in
HT-29 A-2058 A-549 a humidified 5% CO2 atmosphere. The exponentially growing cells were
used throughout the experiments. The MTT assays were performed as
1 1.5 ± 0.3 0.6 ± 0.1 1.1 ± 0.2
2 5.8 ± 0.8 6.7 ± 0.5 7.2 ± 0.8
follows: human cancer cells (1.5–2.5 × 105 cells/mL) were treated with
3 4.1 ± 0.9 4.5 ± 0.9 4.5 ± 1.2 50 μM of each compound for 48 h. Compounds exhibiting cell viabi-
4 3.8 ± 0.4 4.4 ± 1.0 4.1 ± 0.2 lity < 50% were further evaluated at with 1, 10, and 20 μM.
5 15.5 ± 2.7 20.3 ± 5.9 15.6 ± 3.3 Mitoxantrone (Sigma-Aldrich, USA; purity of mitoxantrone was ≥97%)
6 18.5 ± 2.8 23.9 ± 5.0 18.7 ± 4.3
was used at concentrations of 1, 3, 10, and 20 μM as a reference. After
7 5.0 ± 1.2 0.4 ± 0.3 5.7 ± 0.8
MX b
8.7 ± 0.8 10.3 ± 1.1 7.8 ± 0.9 incubation, 0.1 mg (50 μL of a 2 mg/mL solution) MTT (Sigma, Saint
Louis, MO, USA) was added to each well, and the cells were incubated
a
The concentration that inhibits 50% of cell growth was calculated (IC50). at 37 °C for 4 h. The plates were centrifuged at 1000 rpm for 5 min at
Data are means of three experiments. room temperature and the media was then carefully aspirated.
b
Mitoxantrone (MX), an anticancer agent, was used as the reference com- Dimethylsulfoxide (150 μL) was then added to each well to dissolve the
pound. formazan crystals. The plates were read immediately at 540 nm on a
microplate reader (Amersham Pharmacia Biotech., USA). All experi-
300 °C, carrier gas He (2 mL/min). The standard sugars gave peaks at tR ments were performed three times, and the mean absorbance values
(min) 14.11 and 14.26 for D- and L-glucose, respectively, under these were calculated. The results are expressed as the percentage of inhibi-
conditions. Peaks at tR 14.11 min of D-glucose were observed for all tion that produced a reduction in the absorbance by the treatment of
compounds. crude extract or solvent fractions compared to the untreated controls. A
dose-response curve was generated, and the inhibitory concentration of
3.5. Cytotoxic assays 50% (IC50) was determined for each compound and each cell line. The
IC50 values were calculated using Grapad Prism 8.0. The values re-
The effects of 1–7 on the human cancer cells growth were de- present the means ± SD from three independent experiments.
termined by measuring the cytotoxic activity using the 3-[4,5-di-
methylthiazol-2-yl]-2,5-diphenyltetrazolium bromide (MTT) assay.

6
N.S. Thinh, et al.
Funding
This research is funded by Vietnam National Foundation for Science
and Technology Development (NAFOSTED) under grant number
104.01–2015.79 and by the National Research Foundation of Korea
(NRF) grant [NRF-2016R1A2B4006742] funded by the Ministry of
Education, Science and Technology, Republic of Korea.
Declaration of competing interest
No potential conflict of interest was reported by the authors.
Appendix A. Supplementary data
Supplementary data to this article can be found online at https://
doi.org/10.1016/j.phytochem.2019.112255.
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