Cell death-inducing activities via P-glycoprotein inhibition of the constituents isolated from fruits of Nandina domestica

Article abstract of DOI:10.1016/j.fitote.2021.105023

Two new pyrrole alkaloids methyl-E-mangolamide (1) and methyl-Z-mangolamide (2), four new megastigmane glycosides nandinamegastigmanes I–IV (3–6), and eight known compounds (7–14) were isolated from the methanol extract of the fruits of Nandina domestica.

Full text of DOI:10.1016/j.fitote.2021.105023

Fitoterapia 154 (2021) 105023
Contents lists available at ScienceDirect
Fitoterapia
journal homepage: www.elsevier.com/locate/fitote
Cell death-inducing activities via P-glycoprotein inhibition of the
constituents isolated from fruits of Nandina domestica
a
a,
*
b
c
c
Daisuke Imahori , Takahiro Мatsumoto , Youhei Saito , Tomoe Ohta , Tatsusada Yoshida ,
b
a,*
Yuji Nakayama , Tetsushi Watanabe
a
Department of Public Health, Kyoto Pharmaceutical University, Misasagi, Yamashina-ku, Kyoto 607-8412, Japan
Department of Biochemistry & Molecular Biology, Kyoto Pharmaceutical University, Misasagi, Yamashina-ku, Kyoto 607-8412, Japan
Faculty of Pharmaceutical Sciences, Nagasaki International University, Nagasaki 859-3298, Japan
b
c
A R T I C L E I N F O
A B S T R A C T
Keywords:
Two new pyrrole alkaloids methyl-E-mangolamide (1) and methyl-Z-mangolamide (2), four new megastigmane
glycosides nandinamegastigmanes I–IV (3–6), and eight known compounds (7–14) were isolated from the
methanol extract of the fruits of Nandina domestica. The structures of the new compounds were elucidated based
on chemical and spectroscopic evidence. The absolute stereochemistry of nandinamegastigmane I (3) was
established upon comparing the experimental and predicted electronic circular dichroism (ECD) data. Among the
isolated compounds, 1 and 2 showed cell death-inducing activity on the Adriamycin-treated HeLa cells. In
addition, one of the mechanisms for cell death-inducing activity of 1 and 2 was suggested as inhibition of P-
glycoprotein.
Nandina domestica
Nandinamegastigmane
ECD prediction
Adriamycin
Time-lapse cell imaging
P-glycoprotein
1
. Introduction
As a result of long-term and broad research to discover new anti-
the alkaloids from Nandina domestica have cell death-inducing activity
on ADR-treated HeLa cells.
Nandina domestica Thumb. grows wild in Japan and China and be-
longs to the family Berberidaceae which comprises 13 genera and 600
species [10]. In Japan, the fruits of this plant, called Nantenjitsu have
long been used as a folk medicine for treatment of coughs [11]. In China,
they have been used for asthma, whooping cough, pharyngeal tumors,
and uterine bleeding [10,12]. The alkaloids [10] and the flavonoids [12]
have been isolated and identified from the N. domestica. Among them,
aporphine-type alkaloids, such as nantenin is the major active compo-
nent and have been reported to have cytotoxicity. In this study, we
isolated six new constituents from N. domestica and revealed that two
new compounds have cell death-inducing activities under ADR
treatment.
cancer drugs from plants, marine organisms, and microorganisms,
several types of antimutagenic drugs had been reported to show sig-
nificant activity. However, these drugs cause damage to normal tissue
and have critical side effects. In addition, drug resistance occurs through
by DNA repairing [1], mutation of treatment target [2], activation of
detoxifying enzymes [3], anti-apoptotic functions of heat shock protein
(
Hsp) in cancer cells [4], and expression of the drug efflux transporter P-
glycoprotein (P-gp) [5]. For example, Adriamycin (ADR) has been used
as a potent chemotherapeutic drug for the treatment of several cancers
[
6]. ADR induces cell death via producing intercalates with base pairs of
the DNA’s double helix. Although ADR is effective in tumor therapy, the
utilities are limited due to side effects such as cardiotoxicity [7]. The
compounds which enhance the cytotoxicity of ADR such as inhibitors of
P-gp or Hsp, can reduce the dosage of ADR in tumor therapy to avoid the
side effects. According to this background, our research group is inter-
ested in the naturally occurring compounds that enhance the effect of
cancer treatment agents. Previously, we have reported the limonoids
and the guaiane type sesquiterpenoids have cell death-inducing activity
against the ADR-treated cancer cells [8,9]. During our work, we found
2. Results and discussion
2.1. Isolation of constituents from fruits of N. domestica
The methanol extract of the fruits of N. domestica was partitioned in
ethyl acetate–H
2
O (1:1, v/v) to furnish an ethyl acetate fraction and
aqueous layer. Aqueous layer was further partitioned in n-BuOH–H
2
O
*
Corresponding authors.
E-mail addresses: tmatsumo@mb.kyoto-phu.ac.jp (T. Мatsumoto), watanabe@mb.kyoto-phu.ac.jp (T. Watanabe).
https://doi.org/10.1016/j.fitote.2021.105023
Received 6 August 2021; Received in revised form 18 August 2021; Accepted 18 August 2021
Available online 21 August 2021
0
367-326X/© 2021 Elsevier B.V. All rights reserved.

D. Imahori et al.
Fitoterapia 154 (2021) 105023
′
′
′′
(
1:1, v/v) to furnish an BuOH fraction and H
2
O fraction. The ethyl ac-
3 -OMe/C-3 , and H-6-OMe/C-6. These correlations suggested the
etate fraction and BuOH fraction was subjected to normal- and reversed-
phase silica gel column chromatography and high-performance liquid
chromatography (HPLC) to give six new compounds: methyl-E-man-
golamide (1, 0.000036%), methyl-Z-mangolamide (2, 0.00001%),
methoxy group is located at C-6 position and ferulamide moiety is
′
′′
attached C-4 position by amide bond. The geometry of C-7 in 1 and 2
were determined by NOESY spectra and coupling constant of H-NMR
′
′
′′
spectra between H-7 and H-8 (1; 15.2 Hz, 2; 12.4 Hz) as E for 1 and Z
for 2 (Fig. 2). Based on this evidence, the chemical structure of methyl-E-
mangolamide (1) and methyl-Z-mangolamide (2) were determined and
shown in Fig. 1. In these compounds, the naturally occurred 2-formyl-
pyrrole moiety seems to be synthesized via Maillard reaction that is
nonenzymatic reaction of reducing sugars with amines [19].
nandinamegastigmane
.000048%), and IV (6, 0.000071%) together with eight known com-
pounds, (6S,9S)-roseoside (7, 0.000022%) [13], (6S,9R)-roseoside (8,
I (3, 0.00077%), II (4, 0.0008%), III (5,
0
0
0
.000078%) [13], (6R,9R)-9-hydroxy-4-megastigmen-3-one (9,
.000016%) [14], (+)-dehydrovomifoliol (10, 0.00001%) [15], lolio-
lide (11, 0.000056%) [16], 4-O-β-D-glucopyranosylbenzyl-(E)-3-(3,4-
dihydroxyphenyl)acrylate (12, 0.0009%) [17], 4-O-β-D-glucopyr-
anosylbenzyl-(Z)-3-(3,4-dihydroxyphenyl)acrylate (13, 0.00023%)
Nandinamegastigmanes I and II (3 and 4) were isolated as colorless
2
5
amorphous solid with positive optical rotation 3 ([
α
]
D
+ 51.3 in
2
5
MeOH) and 4 ([
α]
D
+ 36.7 in MeOH). The molecular formulas
13
[
17], and 1-p-formylphenyl-6-caffeoyl-β-D-glucopyranoside (14,
(C20
H
32
O
8
) were determined using HRMS and C NMR spectroscopy. A
+
0
.00025%) [17].
molecular ion peak was observed using ESIMS (m/z 423 [M + Na] ).
The 1H and C NMR (CD
13
OD) spectra recorded for 3 and 4 (Table 2)
3
showed signals similar to roseoside [20] except for the signals due to the
atoms around C-9. This analysis and 2D-NMR spectroscopy (Fig. 2)
suggested the nandinamegastigmanes I and II (3 and 4) were the meg-
astigmane glucoside having one more carbon atom at side chain than
roseoside. The geometry of C-7 in 3 and 4 were determined by NOESY
spectra and coupling constant of H-NMR spectra between H-7 and H-8
2
.2. Structures of new compounds (1–6)
Methyl-E-mangolamide (1) and methyl-Z-mango amide (2) were
isolated as colorless amorphous solid. Their molecular formulas
1
3
(
C
21
H
26
N
2
O
5
) were determined using HRMS and C NMR spectroscopy.
A molecular ion peak was observed using EIMS for 1 and 2 (m/z 386
(
1; 15.1 Hz, 2; 9.7 Hz) as E for 3 and Z for 4 (Fig. 2). Subsequently, acid
+
1
13
[
M] ). The H and C NMR (CD
Table 1) show signals corresponding to a magnolamide [18] group {2-
hydroxymethyl-5-formyl pyrrole moiety [1: δ
3
OD) spectra recorded for 1 and 2
hydrolysis of 3 and 4 with 20% aqueous H SO
2
4
–1,4-dioxane yielded D-
(
glucose. D-glucose was identified via HPLC of the chiral tolylth-
iocarbamoyl thiazolidine derivatives of them [21]. The absolute
configuration at C-6 was elucidated from experimental and calculated
ECD spectra (Fig. 3). The experimental ECD spectrum for 3a obtained
via enzymatic hydrolysis from 3 was identical to calculated ECD spec-
trum of 3b. The chemical structure of 3b have been designed for ECD
calculation that assumed to have the same spectra with 3a. The ECD
spectrum of 4a obtained from 4 showed similar cotton effect [242.4 nm
H
6.18 (d, J = 4.1, H-3),
6
.88 (d, J = 4.1, H-4), 4.40 (s, H-6), and 9.35 (s, H-7), 2: δ
H
6.17 (d, J =
4
.1, H-3), 6.88 (d, J = 4.1, H-4), 4.37 (s, H-6), and 9.31 (s, H-7)], 1,4-
′
butanediamine moiety [1: δ
H
4.26 (t-like, J = 7.5, H-1 ), 1.69 (t-like,
′
′
′
J = 7.5, H-2 ), 1.50 (t-like, J = 7.5, H-3 ), and 3.22 (overlapped, H-4 ), 2:
′
′
′
δ
H
4.21 (t-like, J = 7.6, H-1 ), 1.61 (m, H-2 ), 1.44 (m, H-3 ), and 3.21
′
′′
(
m, H-4 )] and ferulamide moiety [1: δ
H
7.02 (d, J = 2.1, H-2 ), 6.69 (d,
′
′
′′
′′
J = 8.3, H-5 ), 6.92 (dd, J = 2.1, 8.3, H-6 ), 7.32 (d, J = 15.2, H-7 ), 6.31
(
Δε +15.5)]. This result suggests that the absolute stereo structure at C-6
′
′
′′
′′
(
d, J = 15.2, H-8 ), and 3.79 (s, H-3 -OMe), 2: δ
H
′′
7.24 (d, J = 2.0, H-2 ),
position of 3 and 4 were S. On the basis of all this evidence, the chemical
structure of nandinamegastigmanes I and II (3 and 4) were determined
and shown in Fig. 1.
′
′
6
7
.61 (d, J = 8.2, H-5 ), 6.82 (dd, J = 2.0, 8.2, H-6 ), 6.51 (d, J = 12.4, H-
′′
′′
), 5.73 (d, J = 12.4, H-7 ), and 3.71 (s, H-3”-OMe)]} with a methoxy
group {1: δ
H
H
3.24 (s, H-6-OMe); 2: δ 3.24 (s, H-6-OMe)}. The positions
Nandinamegastigmane III (5) was isolated as colorless amorphous
2
5
of each functional groups described above were determined based on the
solid with positive optical rotation ([
lecular formulas (C20 ) were determined using HRMS and C NMR
spectroscopy. The 1H and C NMR (CD
Table 2) showed signals similar to nandinamegastigmanes I and II (3
α
]
D
+ 7.4 in MeOH). The mo-
13
HMBC spectra shown in Fig. 2. Namely, long-range correlations were
32 8
H O
′
′
′′
′′
′′
13
observed between following pairs; H-1 /C-5, H-4 /C-9 , H-8 /C-9 , H-
3
OD) spectra recorded for 5
(
Table 1
and 4) except for the signals due to the atoms around C-7, 8, 9, 10, and
1
3
1
C NMR (150 MHz) and H NMR spectroscopic data (600 MHz) of methyl-E-
mangolamide (1) and methyl-Z-mangolamide (2) recorded in CD OD.
11. The NMR spectra show signals due to the sidechain corresponding to
3
a methylene group [δ
H
2.49 (m, H-7a) and 2.65 (overlapping, H-7b)], a
Position
1
2
olefin group [δ
C
124.5 (C-8) and 135.4 (C-9)], a methylene bearing
δ
C
δ
H
(J in Hz)
δ
C
H
δ (J in Hz)
2
3
4
5
6
7
142.0
112.9
126.0
133.8
66.4
140.9
112.9
126.0
133.8
66.4
6.18 (d, 4.1)
6.88 (d, 4.1)
6.17 (d, 4.1)
6.88 (d, 4.1)
4.40 (s)
4.37 (s)
181.1
46.2
9.35 (s)
181.1
46.2
9.31 (s)
E,
E,
Z,
Z,
′
1
4.26 (t-like, 7.5)
1.69 (t-like, 7.5)
1.50 (t-like, 7.5)
3.22 (over lapped solvent
signal)
4.21 (t-like, 7.6)
1.61 (m)
′
2
29.8
29.8
E
Z
′
3
27.7
27.4
1.44 (m)
′
4
40.0
39.9
3.21 (m)
′
′
′
′
′
′
′
′
′
′
′
′
1
2
3
4
5
6
128.2
111.5
149.3
149.9
116.5
123.2
128.5
113.8
148.5
148.5
115.8
124.8
7.02 (d, 2.1)
7.24 (d, 2.0)
E
Z
S
R
6.69 (d, 8.3)
6.61 (d, 8.2)
6.82 (dd, 2.0,
6.92 (dd, 2.1, 8.3)
8
.2)
′
′
′
′
′
′
7
8
9
6
3
141.0
118.7
169.2
58.2
7.32 (d, 15.2)
6.31 (d, 15.2)
138.1
121.7
170.4
58.1
6.51 (d, 12.4)
5.73 (d, 12.4)
-OMe
′
3.24 (s)
3.79 (s)
3.24 (s)
3.71 (s)
Fig. 1. The chemical structures of the constituents (1–14) isolated from fruits
of Nandina domestica.
′
-OMe
56.4
56.4
2

D. Imahori et al.
Fitoterapia 154 (2021) 105023
Table 2
1
3
1
C NMR (150 MHz) and H NMR spectroscopic data (600 MHz) of nandinamegastigmanes I–IV (3–6) recorded in CD
3
OD.
Position
3
4
5
6
δ
C
δ
H
(J in Hz)
δ
C
δ
H
(J in Hz)
δ
C
δ
H
(J in Hz)
δ
C
δ
H
(J in Hz)
1
2
42.5
50.8
42.5
50.1
42.3
50.5
42.7
50.7
α
1.97 (d, 17.2)
α
2.10 (d, 16.5)
α
2.10 (d, 17.2)
α
2.08 (d, 17.2)
β 2.51 (m)
β 2.49 (d, 16.5)
β 2.64 (over lapping)
β 2.39 (d, 17.2)
3
4
5
6
201.8
127.2
167.7
80.4
201.5
127.1
167.7
80.3
201.1
127.2
170.3
79.7
201.2
127.1
167.3
80.4
5.79 (s)
5.87 (s)
5.83 (s)
5.80 (s)
a 2.49 (m)
b 2.65 (m)
5.59 (t, 7.6)
7
131.0
5.54 (d, 15.1)
130.9
5.67 (d, 9.7)
36.5
130.0
5.71 (d, 15.8)
6.26 (d, 15.8)
8
9
135.6
38.2
5.49 (dd, 7.6, 15.1)
2.47 (m)
135.5
38.7
5.68 (m)
124.5
135.4
135.7
137.5
2.58 (m)
a 3.31 (dd, 6.2, 8.9)
b 6.67 (t-like, 8.9)
a 3.77 (dd, 8.9, 6.2)
b 3.44 (dd, 6.8, 8.9)
a 4.01 (d, 8.2)
b 4.17 (d, 8.2)
1
1
0
1
74.9
17.3
75.2
17.3
75.5
14.4
129.4
66.4
5.61 (d, 7.6)
a 4.28 (dd, 7.6, 12.8)
b 4.40 (dd, 7.6, 12.8)
1.73 (s)
0.94 (d, 6.9)
1.05 (d, 6.2)
1.65 (s)
1
1
1
1
1
2
3
4
5
2
3
4
5
’
23.5
24.5
19.8
0.93(s)
0.92(s)
1.82 (s)
23.4
24.5
19.7
1.02 (s)
1.01 (s)
1.91 (s)
24.9
24.2
21.2
0.99 (s)
1.09 (s)
2.00 (s)
13.0
23.5
24.6
19.6
103.2
75.1
78.1
71.7
78.1
0.95 (s)
0.91 (s)
1.81 (s)
104.2
75.2
78.0
71.7
78.2
4.13 (d, 7.6)
3.04 (t, 7.6)
104.4
75.2
78.0
71.7
78.2
4.23 (d, 8.2)
3.15 (t, 8.2)
102.8
75.1
77.9
71.7
78.1
4.17 (d, 8.2)
3.15 (t, 8.2)
4.18 (d, 8.2)
3.08 (t, 8.2)
3.19 (m)
’
’
3.13 (m)
3.26 (m)
3.18 (m)
’
3.15 (m)
3.24 (m)
3.22 (m)
3.17 (m)
’
3.23 (m)
3.32 (m)
3.31 (m)
3.16 (m)
a 3.53 (dd, 4.8, 11.6)
b 3.74 (dd, 1.2, 11.6)
a 3.63 (dd, 5.5, 12.4)
b 3.84 (dd, 1.2, 12.4)
a 3.63 (dd, 5.5, 11.7)
b 3.83 (dd, 1.2, 11.7)
a 3.56 (dd, 5.6, 12.0)
b 3.76 (d-like, 12.0)
6
’
62.8
62.8
62.8
62.8
oxygen function group [δ
H-10b)], and a methyl group [δ
gested that the only difference between 3 and 5 was the location of a
double bond on the side chain. The position of a double bond described
above was determined as C-8/C-9 via HMBC correlations between
following pairs; H-10/C-9, H-11/C-8, H-11/C-9, and H-11/C-10 (Fig. 2).
H
4.01 (d, J = 8.2, H-10a) and 4.17 (d, J = 8.2,
1.65 (s, H-11)]. Above analysis sug-
cytotoxicity was caused by cell death or cell cycle arrest. Therefore, we
performed time-lapse cell imaging analysis using living HeLa cells as
previously reported [8,9,23]. In the control and the tested compounds
(1–14, 3a, and 4a) treated cells, almost all cells progressed to the
mitotic phase within 24 h, whereas the ADR-treated cells did not enter
mitosis (Fig. 5A, B). However, around 70% of cells were not died by ADR
H
Acid hydrolysis of 5 with 20% aqueous H
2
SO
4
–1,4-dioxane yielded D-
treatment at a concentration of 1 g/mL. Interestingly, the compounds 1
μ
glucose same as 3 and 4. Finally, the absolute stereostructure of 5 at C-6
position was deduced as same as 3 and 4. Based on all this evidence, the
chemical structure of nandinamegastigmane III (5) was determined and
shown in Fig. 1.
and 2 significantly increased the number of dead cells under ADR
treatment (p < 0.001) (Fig. 5A, B). Compounds 1 and 2 showed almost
′
′
the same activities; therefore, the geometry of C-7 did not affect the cell
death-inducing activity of ADR. According to the above results, 1 and 2
were selected as potential agents among tested compounds as cell death-
inducing activity of ADR. Among the megastigmane group (3, 3a, 4, and
4a), the sugar moieties were found to decrease the cell death-inducing
activity on ADR treated cells.
Nandinamegastigmane IV (6) was isolated as colorless amorphous
2
5
D
solid with positive optical rotation ([
lecular formulas (C21 ) were determined using HRMS and C NMR
spectroscopy. Via the H, 13C, and 2D NMR (CD
OD) spectra, the
α]
+ 62.8 in MeOH). The mo-
13
32 8
H O
1
3
chemical structure of 6 (Table 2) was elucidated as shown Fig. 1. The
chemical structure of this compound has been described in previous
report however, the chemical structure was deduced only by MS tech-
niques [22]. Therefore, we described several data of this compound such
as NMR and MS spectra in this paper. The chemical structure was
elucidated by same techniques as other new compounds (1–5).
2
.4. Evaluation of the drug efflux activity by P-glycoprotein (P-gp) on
compound 1 and 2 treated HeLa cells
P-gp is a member of the human ATP-binding cassette (ABC) family
and causes drug resistance in cancer cells [24,25]. These ABC trans-
porters have been suggested to transport anticancer drugs, including
Anthracyclines [26], Vinca alkaloids [27], and some molecular targeted
drugs [28] such as imatinib, erlotinib, and sunitinib. Epidemiological
studies have shown that MDR 1 gene is overexpressed in many tumors
[29]. Therefore, the overexpression of the ABC transporter has been
linked to clinical multidrug resistance in solid tumors and blood cancers,
which remains a significant obstacle to successful cancer chemotherapy
[30]. The HeLa cells expressing P-gp and it relates to drug resistance
[31]. We further examined the inhibitory effects of 1 and 2 against P-gp
in HeLa cells by Operetta high-content imaging system. We used
Rhodamine 123 (Rh 123) as an artificial substrate of P-gp [32] and the
amounts of Rh 123 in living cells were evaluated by fluorescence in-
tensity. For positive control, verapamil was used as P-gp inhibitor [33].
In the control group, after co-culture with Rh 123, relative fluorescence
intensity was decreased to 58.9% (3 h) and 24.1% (6 h). On the other
hand, verapamil, 1, and 2 suppressed the decrement of relative fluo-
rescence intensity as 99.0% (verapamil), 67.3% (1), and 57.9% (2) of
2
.3. Evaluation cytotoxicity and cell death analysis
The cytotoxicity of the isolated compounds (1–14) and derivatives
3a and 4a) were evaluated in human cervical cancer (HeLa) cells with
(
or without ADR. The cell viability was examined using crystal violet
staining and WST-8 assay. In this assay, tested compounds (1–14, 3a,
and 4a) did not show cytotoxicity at 60 M for 24 h (Fig. 4A). However,
μ
combination treatment of 1 and 2 (60
stronger cytotoxicity than that of the only ADR (1
the crystal violet staining and WST-8 assay (Fig. 4B, C). On the WST-8
assay, IC50 value of ADR (IC50 of ADR: 1.69 ± 0.11 M) was signifi-
cantly decreased by treatment of 1 (IC50 of ADR: 0.72 ± 0.04 M) or 2
M) (p < 0.001) (Fig. 4D, E). These results
μ
M) with ADR (1
μ
g/mL) showed
μ
g/mL) treatment on
μ
μ
(
IC50 of ADR: 0.65 ± 0.08 μ
suggested that the compounds 1 and 2 may be able to decrease the
amount of ADR for cancer treatment.
Only using cell viability assay, it is difficult to evaluate whether the
3

D. Imahori et al.
Fitoterapia 154 (2021) 105023
Fig. 2. The important 2D NMR and NOESY correlations for new compounds (1–6).
the intensities were still observed after 6 h, resulting that the Rh 123
3. Experimental section
efflux inhibition rate was 98.7% (verapamil), 56.9% (1), and 44.6% (2)
(
Fig. 6). In these experiments, test compounds did not affect the cell
3.1. General experimental procedures
proliferation at 30 M and 60 M. These results suggests that the inhi-
μ
μ
bition of P-gp activity may be one of the mechanisms of cell death-
Specific rotations were obtained on a JASCO P-2200 digital polar-
imeter (l = 5 cm) (JASCO, Tokyo, Japan). FTIR spectra were recorded on
a JASCO FT/IR-4600 Fourier transform infrared spectrometer (JASCO).
ECD spectroscopy was recorded on a JASCO J-1500 spectrometer
inducing activity of 1 and 2 on ADR-treated cells.
4

D. Imahori et al.
Fitoterapia 154 (2021) 105023
ent
(S)
(R)
ent
Fig. 3. A comparison of the experimental (3a) and calculated (3b and ent-3b)
ECD spectra.
Fig. 5. The time lapse imaging analysis of the isolated compounds (1–14) and
derivatives (3a and 4a): (A, B) HeLa cells treated with 60 μM of the indicated
compounds or 0.5, 1.0, 2.0
changes in the cell morphology were determined using time-lapse recording.
A) The percentages of mitotic entry cells (white columns) or dead cells (red
μ
g/mL of ADR for 24 h. During these treatments, the
(
columns) are reported as the mean ± S.D. of three independent experiments.
Statistical significance was analyzed using the Tukey–Kramer test. (***P <
0
.001 compared with 1 g/mL of ADR-treated cells). (B) Representative images
μ
are shown. White arrowheads indicate the mitotic cells and red arrowheads
indicate dead cells. (For interpretation of the references to colour in this figure
legend, the reader is referred to the web version of this article.)
Fig. 4. The cytotoxic effects of the isolated compounds (1–14) and derivatives
(
3a and 4a): (A–E) (A) HeLa cells were treated with 60
μ
M of the indicated
compounds or 0.5, 1, 2
staining. ADR was used as a positive control. (B) Crystal violet staining of HeLa
cells treated with 60 M of the indicated compounds in combination with ADR
μ
g/mL of ADR for 24 h and subjected to crystal violet
(
600 MHz) spectrometer (JEOL).
Normal-phase silica gel column chromatography was carried out
μ
using Silica gel 60 (63–210 mesh) (Kanto Chemical, Tokyo, Japan) and
for 24 h. (C) The percentage cell viability measured by crystal violet staining
was reported as the mean ± SD of three independent experiments. (D, E) HeLa
reversed-phase silica gel column chromatography was carried out on
◦
C18-OPN (140 m) (Nacalai Tesque, Kyoto, Japan). Thin-layer chro-
μ
cells were seeded into 96-well plates (3000 cells/well) and cultured at 37 C.
After 24 h, ADR was added to the culture medium at varying concentrations of
matography (TLC) was performed using TLC plates pre-coated with
60F254 silica gel (0.25 mm; ordinary phase) (Merck, Darmstadt, Ger-
many) and RP-18 F254S silica gel (0.25 mm; reversed-phase) (Merck).
Reversed-phase high-performance TLC was carried out using TLC plates
pre-coated with RP-18 WF254S silica gel (0.25 mm) (Merck). HPLC was
performed using a Shimadzu SPD-M10Avp UV–vis detector (Shimadzu).
COSMOSIL 5C18-MS-II (250 × 4.6 mm i.d. and 250 × 20 mm i.d.)
0
–2.5 ◦
μ
M with or without 60 μM of compounds 1 and 2, and cells were cultured
at 37 C for 24 h. (D) Surviving cells were detected by WST-8 assay. Data are
expressed as means ± S.D. in triplicate experiments. Data are representative of
at least three independent experiments. (E) IC50 values are expressed as mean
±
S.D. Statistical significance was analyzed using the Tukey–Kramer test. (***P
0.001 compared with DMSO-treated cells). (For interpretation of the refer-
<
ences to colour in this figure legend, the reader is referred to the web version of
this article.)
(
Nacalai Tesque) and YMC-Triart PFP (250 × 4.6 mm i.d. and 250 × 20
mm i.d.) (YMC, Kyoto, Japan) columns were used for analytical and
preparative purposes.
(
JASCO). ESIMS and high-resolution ESIMS was recorded on a Shimadzu
LCMS-IT-TOF (Shimadzu, Kyoto, Japan). EIMS and HREIMS were
recorded on JEOL JMS-GCMATE mass spectrometer (JEOL, Tokyo,
3
.2. Plant material
1
Japan). H NMR spectroscopy was recorded on JEOL ECS400 (400 MHz)
and JNM-ECA 600 (600 MHz) spectrometers (JEOL). 13C NMR spec-
troscopy was recorded on a JNM-ECA 600 (150 MHz) spectrometer
The fruits of N. domestica distributed in the Nara prefecture of Japan
were purchased from Tochimoto tenkaido (Osaka prefecture, Japan) in
November 2019.
(
JEOL). 2D-NMR experiments were carried out on a JEOL JNM-ECA 600
5

D. Imahori et al.
Fitoterapia 154 (2021) 105023
B6–4 (474.2 mg) was purified using HPLC {H
give 3 (20.8 mg), 4 (21.6 mg), 5 (1.3 mg), and 6 (1.9 mg). Fraction B7
17.3 g) was separated using reversed-phase silica gel column chroma-
tography to give nine fractions. Fraction B7–4 (223.4 mg) was purified
using HPLC {H CN (80:20, v/v)} to give 12 (24.1 mg), 13 (6.2
O–CH
mg), and 14 (6.6 mg).
2
O–CH
3
CN (85:15, v/v)} to
(
2
3
3
.4. Methyl-E-mangolamide (1)
Amorphous powder; 1H NMR (CD
CD
OD, 600 MHz) and 13C NMR
3
+
(
3
OD, 150 MHz), see Table 1; EIMS m/z 386 [M] ; HREIMS m/z
3
86.1845 [M + Na]+ (calcd for C21
26 2 5
H N O , 386.1842).
3
.5. Methyl-Z-mangolamide (2)
Amorphous powder; 1H NMR (CD
CD
OD, 600 MHz) and 13C NMR
3
+
(
3
OD, 150 MHz), see Table 1; EIMS m/z 386 [M] ; HREIMS m/z
3
86.1841 [M + Na]+ (calcd for C21
H
26
N
2
O
5
, 386.1842).
3
.6. Nandinamegastigmane I (3)
1
Amorphous powder; [
α
]25
D
+ 51.3 (c 0.1, MeOH); H NMR (CD
3
OD,
00 MHz) and 1 C NMR (CD
3
OD, 150 MHz), see Table 2; ESIMS m/z 423
6
3
+
Na [M + Na]+,
[
M + Na] ; HRESIMS m/z 423.1994 (calcd for C20
H
32
O
8
4
23.1989).
3
.7. Nandinamegastigmane II (4)
Fig. 6. Effects of isolated compound 1 and 2 on the efflux inhibition of Rh 123:
(
A) HeLa cells were incubated with Rh 123 (1
μ
M) for 2 h. After removal of
]25
1
Amorphous powder; [
α
D
+ 36.7 (c 0.1, MeOH); H NMR (CD
3
OD,
Rh123, cells were treated with verapamil (30 M), compounds 1 and 2 (60
μ
μM)
3
6
00 MHz) and 1 C NMR (CD
3
OD, 150 MHz), see Table 2; ESIMS m/z 423
or DMSO for 6 h, and fluorescence was detected by Operetta cell imaging
system. Data are representative of at three independent experiments. (B) The
percentages of Rh 123 fluorescence are shown as means ± S.D. of three inde-
pendent experiments. The intensity of fluorescence was quantified at the same
field at 0–6 h as described in the “Experimental section”. Each graph was
normalized with the 0-h time point as 100%. Statistical significance was
analyzed using Dunnett’s test (**P < 0.01 compared with DMSO-treated cells).
+
Na [M + Na]+,
[
M + Na] ; HRESIMS m/z 423.1991 (calcd for C20
32 8
H O
4
23.1989).
3
.8. Nandinamegastigmane III (5)
]25
1
Amorphous powder; [
α
D
+ 7.4 (c 0.3, MeOH); H NMR (CD
3
OD,
6
00 MHz) and 13C NMR (CD
3
OD, 150 MHz), see Table 2; ECD: Δε
(nm)
3
.3. Extraction and isolation
-
2.80 (199.4), +0.52 (214.0), ꢀ 0.18 (234.7), +0.49 (268.1) (MeOH);
+
ESIMS m/z 423 [M + Na] ; HRESIMS m/z 423.1984 (calcd for
The dried fruits of N. domestica (5 kg) were extracted three times with
+
C
20
H
32
O
8
Na [M + Na] , 423.1989).
methanol under reflux for 3 h. Evaporation of the solvent provided a
methanol extract (1160 g, 23.2%). The methanol extract was partitioned
into ethyl acetate–water (1:1, v/v) to furnish an ethyl acetate fraction
3
.9. Nandinamegastigmane IV (6)
(
40.6 g, 0.81%) and water-soluble fraction. The water-soluble fraction
was further partitioned into n-BuOH–water (1:1, v/v) to furnish a BuOH
fraction (111.7 g, 2.2%) and H O fraction. The ethyl acetate-soluble
fraction was subjected to normal-phase silica gel column chromatog-
raphy [1200 g, n-hexane–CHCl (1:1 → 1:5, v/v) → CHCl –MeOH (1:0
1
Amorphous powder; [
α
]25
D
+ 62.8 (c 0.3, MeOH); H NMR (CD
3
OD,
6
00 MHz) and 13C NMR (CD
3
OD, 150 MHz), see Table 2; ECD: Δ
ε
(nm)
2
+
-
2.2 (221.7), +2.4 (250.3) (MeOH); ESIMS m/z 435 [M + Na] ; HRE-
+
SIMS m/z 435.1984 (calcd for C21
H
32
O
8
Na [M + Na] , 435.1989).
3
3
→
50:1 → 20:1 → 10:1 → 1:1, v/v)] to give eight fractions (EA1–EA8).
3
.10. Acid hydrolysis of nandinamegastigmanes I–IV (3–6)
Fraction EA4 (1.4 g) was separated using reversed-phase silica gel col-
umn chromatography to give seven fractions. Fraction EA4–3 (72.5 mg)
The solution of 3–6 (each 0.5 mg) in 20% aqueous H
2
SO
4
–1,4-
was purified using HPLC {H
2
O–CH
3
CN (80:20, v/v)} to give 10 (5.2 mg)
◦
dioxane were heated 90 C and stirred for 2 h with a breflux condenser.
and 11 (2.8 mg). Fraction EA4–4 (36.6 mg) was purified using HPLC
CN (80:20, v/v)} to give 9 (0.8 mg). Fraction EA5 (3.0 g) was
After the acid hydrolysis of 3–6, solutions were neutralized and
extracted with EtOAc and H O. After drying in vacuo, H O soluble
2 2
{
H
2
O–CH
3
separated using reversed-phase silica gel column chromatography to
fraction was dissolved in pyridine (0.1 mL) containing L-cysteine methyl
give nine fractions. Fraction EA5–4 (163.6 mg) was purified using HPLC
◦
ester hydrochloride (0.5 mg) and heated at 60 C for 1 h. A solution of o-
{
H
2
O–CH
3
CN–CH
3
COOH (65:35:0.3, v/v/v)} to give 1 (1.8 mg) and 2
tolylisothiocyanate (0.5 mg) in pyridine (0.1 mL) was added, and the
(
0.5 mg). The part of BuOH fraction (60.0 g) was subjected to normal-
◦
mixture was heated at 60 C for 1 h. The mixture was analyzed by
phase silica gel column chromatography [1200 g, n-hexane–CHCl
3
reversed-phase HPLC [column: COSMOSIL 5C18-AR-II, 250 × 4.6 mm i.
(
1:1 → 1:4, v/v) → CHCl
3
–MeOH (1:0 → 50:1 → 40:1 → 30:1 → 20:1 →
d. (5
μ
m); mobile phase: 20% CH
3
CN in 50 mM H
3
PO
4
; detection: UV
1
0:1 → 1:1, v/v)] to give eight fractions (B1–B8). Fraction B6 (5.8 g) was
◦
(
254 nm); flow rate: 1.0 mL/min; column temperature: 25 C] to identify
separated using reversed-phase silica gel column chromatography to
the derivatives of constituent D-glucose by comparison of their retention
times with those of authentic samples (RT: D-glucose, 37.5 min; L-
glucose, 29.9 min).
give ten fractions. Fraction B6–3 (383.3 mg) was purified using HPLC
2
{
H
O–CH
3
CN (90:10, v/v)} to give 7 (0.6 mg) and 8 (2.1 mg). Fraction
6

D. Imahori et al.
Fitoterapia 154 (2021) 105023
3
.11. Enzymatic hydrolysis of nandinamegastigmanes I and II (3 and 4)
The solution of 3 or 4 (each 5 mg) in 20 mM acetate buffer (3.0 mL,
were solubilized in 100
μ
L methanol and absorbance was detected at
5
95 nm using a microplate reader (Sunrise Thermo RC-R; Tecan Austria
GmbH, Grodig, Austria).
pH = 5.0) were added β-glucosidase (2 mg, from Sweet armond) and the
◦
mixtures were stirred for 24 h at 37 C. The supernatant solutions were
3.17. WST-8 assay
concentrated under vacuum to give the residues, which were subjected
to HPLC {COSMOSIL 5C18-MS-II, H
1.3 mg) or 4a (0.8 mg).
2
O–CH
3
CN (70:30, v/v)} to give 3a
Cell viability was determined using a cell counting kit 8 (CCK-8;
Dojindo, Kumamoto, Japan) method as described previously [8].
Briefly, cells were seeded in 96-well cell culture plates. After approxi-
mately 24 h, the cells were treated with ADR (Wako Pure Chemical
(
3
.12. Aglycone of 3 (3a)
Industries) with or without isolated compounds (60 M) for 24 h. The
μ
Amorphous powder; [
α
]25
D
+ 64.7 (c 0.26, MeOH); H NMR (CDCl
1
3
,
absorbance was measured at 450 and 650 nm using a microplate reader.
Values for the concentration that inhibited growth by 50% (IC50) were
calculated using GraphPad Prism 8.43 (GraphPad Software, San Diego,
CA, USA).
6
4
3
1
4
7
00 MHz,) δ 2.22 (d, J = 17.2, H-2), 2.43 (d, J = 17.2, H-2), 5.91 (s, H-
), 5.62 (d, J = 15.8, H-7), 5.66 (dd, J = 15.8, 6.8, H-8), 2.45 (m, H-9),
.47 (m, H-10), 3.54 (m, H-10), 1.04 (d, J = 6.9, H-11), 1.08 (s, H-12),
.01 (s, H-13), 1.90 (s, H-14); 13C NMR (CDCl
, 150 MHz); δ 41.1 (C-1),
3
9.8 (C-2), 198.1 (C-3), 126.9 (C-4), 162.9 (C-5), 79.3 (C-6), 130.4 (C-
3.18. Time-lapse imaging
), 134.3 (C-8), 39.4 (C-9), 67.3 (C-10), 16.5 (C-11), 22.9 (C-12), 24.1
+
(
C-13), 19.0 (C-14); EIMS m/z 238 [M] ; HREIMS m/z 238.1568 (calcd
Time-lapse imaging was performed on an Operetta high-content
imaging system (PerkinElmer, Waltham, MA, USA) as described previ-
ously [8,9,21]. Briefly, the cells were cultured in a flat-bottomed 96-well
plate (Coster 3596; Corning, NY, USA) to reach 70–80% confluence. The
cells were then treated with compound 1–14 and ADR just prior to the
+
for C14
H
22
O
3
[M] , 238.1569).
3
.13. Aglycone of 4 (4a)
Amorphous powder; [
1
α
]25
D
+ 63.7 (c 0.5, MeOH); H NMR (CDCl
3
,
time-lapse cell imaging. The images were captured at 10 min intervals
◦
6
4
3
1
1
00 MHz,) δ 2.22 (d, J = 17.2, H-2), 2.43 (d, J = 17.2, H-2), 5.91 (s, H-
), 5.64 (d, J = 6.0, H-7), 5.64 (m, H-8), 3.48 (m, H-9), 3.47 (m, H-10),
.55 (m, H-10), 1.02 (d, J = 6.9, H-11), 1.08 (s, H-12), 1.02 (s, H-13),
for 24 h under a 5% CO
2
atmosphere at 37 C.
3.19. Evaluation of the activity of P-glycoprotein on HeLa cells
.90 (s, H-14); 13C NMR (CDCl
, 150 MHz); δ 41.1 (C-1), 49.7 (C-2),
3
98.1 (C-3), 126.9 (C-4), missing (C-5), 79.3 (C-6), 130.5 (C-7), 134.3
In this assay, we observed the green fluorescence of Rh 123 (Wako
Pure Chemical Industries) to evaluate the efflux activity of P-gp. The
exponentially growing HeLa cells were seeded into 24-well plates
(
C-8), 39.6 (C-9), 67.3 (C-10), 16.6 (C-11), 22.9 (C-12), 24.1 (C-13),
+
1
8.9 (C-14); ESIMS m/z 239 [M] ; HRESIMS m/z 239.1617 (calcd for
+
4
C
14
H
23
O
3
[M + H] , 239.1642).
(Coster 3526; Corning) at a density of 8 × 10 cells/mL in a final volume
of 500 L medium for 12 h. After incubation, the culture medium was
μ
3
.14. Calculation of the theoretical ECD spectra of 3b
replaced to medium containing Rh 123 (1
cultured in the dark for 2 h under a 5% CO
μ
M), and the cells were
◦
2
atmosphere at 37 C. Then,
The initial geometries of conformers for enantiomer, 3b (6S) and ent-
the cells were washed with fresh ice-cold medium three times to remove
non-absorbed Rh 123 and incubated with or without 60 M of individual
3
b (6R), were generated and then geometrically optimized in vacuum by
μ
using the Merck molecular force field (MMFF) as implemented in
Spartan ‘10 program [34]. The initial low-energy conformers for each
enantiomer with Boltzmann distributions over 1% were further opti-
mized and verified stability at the CAM-B3LYP/6–31G(d) level of long-
range corrected density functional theory (DFT). The 20 low-energy
conformers for each enantiomer with Boltzmann distributions over 1%
were subjected to the ECD calculations using time-dependent density
functional theory (TD-DFT) at the CAM-B3LYP/def2-TZVP level. Ge-
ometry optimizations and ECD calculations were both carried out with
an integral equation formalism polarizable continuum model (IEFPCM)
in MeOH using Gaussian 16 program [35]. The calculated ECD curves
were generated using SpecDis v1.71 [36].
isolated compounds (1 and 2) and verapamil (V4629; Sigma-Aldrich) as
a positive control just prior to the time-lapse cell imaging. The images
were captured at 30 min intervals for 6 h under a 5% CO
2
atmosphere at
◦
37 C. Fluorescence images were obtained with an Operetta high-
content imaging system (PerkinElmer). The excitation and emission
wavelengths were 460–490 nm and 500–550 nm, respectively. The
images were observed and examined using 20 × objective. Lamp power
and exposure time were set and kept constant throughout each experi-
ment to avoid overexposure of the fluorescence signal. Figures were
prepared with Photoshop 2021 (Adobe, San Jose, CA, USA) and Illus-
trator 2021 (Adobe). Integrated fluorescence intensity was determined
by ImageJ software (NIH, USA) from the images of three independent
experiments.
3
.15. Cells
3
.20. Statistical analysis
Human cervical carcinoma (HeLa) cells were maintained in Dul-
becco’s modified Eagle’s medium (DMEM) with low glucose (Wako Pure
Statistical analyses were performed using GraphPad Prism 8.43
Chemical Industries, Osaka, Japan) supplemented with 5% fetal bovine
software. The statistical analysis was conducted using one-way analysis
of variance (ANOVA) followed by a Tukey-Kramer or Dunnett’s test to
analyze the differences between the treatment groups. The differences
were considered significant when ***P < 0.001 or**P < 0.01.
serum (Sigma-Aldrich, St Louis, MO, USA) under a 5% CO
2
atmosphere
◦
at 37 C.
3
.16. Crystal violet staining
Contribution
Crystal violet staining was performed described previously [8].
Briefly, cells were fixed and stained with 100 L of 50% methanol
containing 0.5% (v/v) crystal violet solution (V5265; Sigma-Aldrich) for
min. After fixation and staining, the mixture was removed and washed
μ
onceptualization: T. Matsumoto; Data curation: D. Imahori, T. Mat-
sumoto, Y. Saito, T. Ohta; Formal analysis, Visualization, Investigation,
Validation, and Writing - original draft: D. Imahori, T. Matsumoto;
Funding acquisition: T. Matsumoto; Methodology: T. Matsumoto, Y.
Saito; Project administration: Y. Nakayama, T. Watanabe; Resources and
5
with tap water. The plates were dried at room temperature. Cell viability
was measured following crystal violet staining. Fixed and stained cells
7

D. Imahori et al.
Fitoterapia 154 (2021) 105023
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Declaration of Competing Interest
[
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