Cell death-inducing activities via Hsp inhibition of the sesquiterpenes isolated from Valeriana fauriei

Article abstract of DOI:10.1007/s11418-021-01543-9

Three new sesquiterpenes, valerianaterpenes I–III, and eight known compounds have been isolated from the methanol extract of the rhizomes and roots of Valeriana fauriei. The chemical structures of the three new sesquiterpenes were elucidated based on chem

Full text of DOI:10.1007/s11418-021-01543-9

Journal of Natural Medicines
https://doi.org/10.1007/s11418-021-01543-9
ORIGINAL PAPER
Cell death‑inducing activities via Hsp inhibition of the sesquiterpenes
isolated from Valeriana fauriei
Takahiro Matsumoto1 · Takahiro Kitagawa1 · Daisuke Imahori1 · Hayato Yoshikawa1 · Masaya Okayama1 ·
Mayuka Kobayashi1 · Naoto Kojima1 · Masayuki Yamashita1 · Tetsushi Watanabe1
Received: 26 May 2021 / Accepted: 25 June 2021
© The Japanese Society of Pharmacognosy 2021
Abstract
Three new sesquiterpenes, valerianaterpenes I–III, and eight known compounds have been isolated from the methanol extract
of the rhizomes and roots of Valeriana fauriei. The chemical structures of the three new sesquiterpenes were elucidated based
on chemical and spectroscopic evidence. The absolute stereochemistry of valerianaterpene I was determined using X-ray
crystallography. The cell death-inducing activity of isolated compounds alone or combination with Adriamycin (ADR) was
observed by time-lapse cell imaging. Although the isolated compounds did not afect the number of mitotic entry cells and
dead cells alone, kessyl glycol, kessyl glycol diacetate, and iso-teucladiol signifcantly increased the number of dead cells
on ADR treated human cervical cancer cells. One of the mechanisms of cell death-inducing activity for the kessyl glycol
acetate was suggested to be the inhibition of heat-shock protein 105 (Hsp105) expression level. This paper frst deals with
the naturally occurring compounds as Hsp105 inhibitor.
Keywords Valeriana fauriei · Valerianaterpene · X-ray crystallography · Time-lapse cell imaging · Heat-shock protein
Introduction
there are quite few biological investigations for its charac-
teristic constituents such as α-kessyl alcohol derivatives that
have cyclized guaiane-type structures [9]. On our ongoing
research program for the discovery of new cancer treatments
and prevention agents [10–13], we pursued the isolation of
characteristic sesquiterpenes enhancing the cytotoxicity of
adriamycin (ADR) from V. fauriei.
The genus Valeriana (Valerianaceae) contained more than
250 species of plants has been used as a mild sedative and
sleep aid agent in Europe, Asia, and North America [1].
Among them, Valeriana ofcinalis is the ofcial species in
the European Pharmacopoeia and is commonly referred to
as valerian [2]. From V. ofcinalis, iridoids [3] and sesquit-
erpenoids [4] including rare N-containing bisesquiterpenoid
derivative [5] had been reported. On the other hand, Valeri-
ana fauriei Briq., which belongs to the same genus plant as
V. ofcinalis L. is abundant in the east Asia countries such
as Japan and China, has been used for hundreds of years in
folk medicine [6]. From V. fauriei, iridoids [7] and sesquit-
erpenoids [8] were reported same as V. ofcinalis. However,
number of references about phytochemical research for V.
fauriei is more limited than that of V. ofcinalis. In addition,
ADR has been used as a potent chemotherapeutic drug
for the treatment of several cancers [14]. ADR induces
cell death via producing intercalates with base pairs of the
DNA’s double helix. Although this is efective in tumor ther-
apy, the utilities of this compound are limited due to cardio-
toxicity which is known to side efects [15]. Therefore, the
compounds enhancing the cytotoxicity of ADR can reduce
the dosage of ADR in tumor therapy to avoid the side efects
should have the potency of new cancer treatment drugs. In
addition, the anti-apoptotic function of heat-shock proteins
(Hsps) had been suggested to be one of the major mecha-
nisms on ADR registration of cancer cell [16]. Therefore, we
evaluated inhibitory efects against the expression levels of
several Hsp for cell death-induced compound.
* Takahiro Matsumoto
tmatsumo@mb.kyoto-phu.ac.jp
* Tetsushi Watanabe
watanabe@mb.kyoto-phu.ac.jp
1
Kyoto Pharmaceutical University, Misasagi, Yamashina-ku,
Kyoto 607-8412, Japan
Vol.:(0123456789)
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Journal of Natural Medicines
Results and discussion
The methanol extract of the rhizomes and roots of V. fauriei
was partitioned in ethyl acetate–H2O (1:1, v/v) to furnish
an ethyl acetate-soluble fraction (3.1%) and aqueous layer
(8.0%). The ethyl acetate-soluble fraction was subjected to
normal- and reversed-phase silica gel column chromatogra-
phy and repeated high-performance liquid chromatography
(HPLC) to give three new compounds: valerianaterpene I (1,
0.0003%), II (5, 0.00095%), and III (10, 0.00031%), together
with eight known compounds, 7β-hydroperoxy eudesma-
11-en-4-ol (2, 0.00042%) [17], kanokonol (3, 0.0032%) [6],
15-acetoxyvaleranone (4, 0.018%) [18], α-kessyl alcohol (6,
0.00032%) [6], kessyl glycol (7, 0.0052%) [9], kessyl glycol
acetate (8, 0.0091%) [9], kessyl glycol diacetate (9, 0.26%)
[9], and iso-teucladiol (11, 0.26%) [19] (Fig. 1).
Valerianaterpene I (1) was isolated as colorless needle
crystals (1) with positive optical rotations ([α]25D + 8.8 in
MeOH). Its molecular formula (C17H28O3) was determined
using HRMS and 13C NMR spectroscopy. A molecular ion
peak was observed using EIMS for 1 (m/z 280 [M]+). The
1H and 13C NMR (CDCl3) spectra recorded for 1 (Table 1)
show signals corresponding to a maaliol [20] moiety {three
Fig. 1 The chemical structures of the isolated constituents (1–11)
from V. fauriei
Table 1 13C NMR (150 MHz) and 1H NMR spectroscopic data (600 MHz) of valerianaterpenes I–III (1, 5, and 10) and 5a recorded in CDCl3
Position
1
5
5a
10
δC
δH (J in Hz)
δC
δH (J in Hz)
δC
δH (J in Hz)
δC
δH (J in Hz)
1
2
3
34.0
19.9
42.4
α 0.82 (m)
73.5
43.2
30.7
4.17 (t-like, 4.1)
73.8 4.08 (m)
27.6
40.6
81.1
α 1.55 (m)
β 1.64 (m)
β 1.75 (m)
1.47 (m)
α 1.33 (ddd, 8.9, 8.9, 15.1)
43.4 α 1.26 (m)
α 1.77 (m)
β 2.03 (dd-like, 8.9, 15.1)
β 1.29 (m)
β 1.69 (m)
α 1.86 (m)
2.28 (m)
2.94 (m)
30.4 2.22 (m)
35.8 2.75 (m)
β 1.39 (m)
4
5
72.3
50.1
35.9
26.5
52.5
25.5
1.75 (m)
1.67 (m)
1.14 (d, 6.8)
α 1.58 (ddd, 7.6, 14.4, 14.4) 29.9 α 2.01 (m)
β 2.12 (ddd, 7.6, 14.4, 14.4)
2.33 (d-like, 7.6)
β 1.99 (m)
46.5 1.67 (d-like, 6.0)
73.1 4.06 (m)
6
7
19.2
19.6
0.50 (dd, 6.8, 9.6) 54.8
0.70 (t-like, 8.2)
47.4
27.0
1.75 (m)
213.0
52.2
74.3
α 1.28 (m)
β 1.87 (m)
8
9
15.5
34.5
α 1.73 (m)
α 2.36 (d, 18.5)
45.5 α 2.16 (dd, 9.6, 13.8) 37.9
α 2.04 (m)
β 1.53 (m)
β 2.67 (d, 18.5)
β 1.87 (dd, 8.4, 13.8)
β 2.54 (ddd, 2.8, 6.9, 13.7)
α 1.64 (m)
72.5
153.3
β 0.61 (m)
10
11
12
13
14
15
36.7
18.5
15.5
29.1
23.8
63.3
55.4
75.5
27.3
33.0
17.6
27.0
1.43 (dd, 3.2, 13.6)
53.9 1.50 (m)
75.8
28.8 1.31 (s)
32.3 1.40 (s)
18.1 0.79 (d, 7.2)
27.7 1.44 (s)
48.2
74.2
27.2
26.7
23.8
2.20 (dd, 8.0, 17.6)
0.97 (s)
1.04 (s)
1.26 (s)
1.54 (s)
1.23 (s)
0.76 (d, 6.8)
1.42 (s)
1.19 (s)
1.17 (s)
1.21 (s)
4.06 (d, 13.0)
106.4 4.69 (br-s)
4.27 (d, 13.0)
OAc
171.5
21.0
2.04 (s)
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Journal of Natural Medicines
methyl groups [δH0.97 (s, H-12), 1.04 (s, H-13), and 1.26 (s,
H-14)], a methylene bearing oxygen function group [δH 4.06
(d, J=13.0, H-15) and 4.27 (d, J=13.0, H-15)], fve methyl-
enes [δC 34.0 (C-1), 19.9 (C-2), 42.4 (C-3), 15.5 (C-8), and
34.5 (C-9)], a methyne [δH 1.14 (d, J=6.8, H-5), a quater-
nary carbon bearing oxygen function group [δC 72.3 (C-4)],
and a quaternary carbon [δC 36.7 (C-10)]} together with an
acetyl group [δC 171.5 (OCOCH3) and 21.0 (OCOCH3)].
The positions of the acyl group described above were deter-
mined based on the DQF COSY and HMBC spectra shown
in Fig. 2. Namely, the long-range correlations were observed
between H-5/C-15 and H-15/C-1, 9, 10, and OCOCH3 sug-
gested the acyl group was attached at C-15 (Fig. 2). For-
tunately, crystals of 1 were obtained from MeOH–H2O
and X-ray crystallography used to determine the absolute
stereochemistry of 1 (Fig. 3). Namely, the absolute stereo-
chemistry was determined to be 4R,5S,6R,7R,10R using the
Flack parameter [absolute structure parameter= 0.05(9)].
The NOESY spectra suggested above confguration same
as X-ray crystallography data. Based on this evidence, the
chemical structure of valerianaterpene I (1) was determined
and shown in Fig. 1.
Fig. 3 ORTEP structure of valerianaterpene I (1)
show signals similar to kessyl glycol (7) including four
methyl groups [δH 1.54 (s, H-12), 1.23 (s, H-13), 0.76 (d,
J=6.8, H-14), and 1.42 (s, H-15)], and two quaternary car-
bons bearing oxygen function group [δC74.3 (C-9) and 75.5
(C-11)]. The diference of 5 from 7 was the disappearance
of a methine bearing oxygen function group at C-7 position
and appearance of a carbonyl group. The position of car-
bonyl group described above was determined as C-7 from
HMBC correlations between H-8/C-7 and H-6/C-7 (Fig. 2).
NOESY cross-peaks corresponding to H-1/H-10, H-1/H-
2α, H-10/H-12, and H-2α/H-14 suggested that H-1, H-2α,
H-10, H-12, H-13, and H-14 were located on same side. In
addition, NOESY cross-peaks corresponding to H-2β/H-3,
H-2 β/H-4, H-4/H-5β, H-5β/H-6, H-4/H-15 suggested that
H-2β, H-3, H-4, H-5β, H-6, and H-15 were located on same
side. Therefore, the relative structure of 5 was determined
as 1R*,3R*,4R*,6R*,9S*,10S*. Finally, the absolute stereo-
chemistry of 5 was determined as 1R,3R,4R,6R,9S,10S from
derivatization to kessyl glycol (7). Namely, reduction of 3
using NaBH4 obtained 7 together with 5a. Synthesized 7
was identifed via 1H NMR, MS, and optical rotation sug-
gested absolute stereochemistry of 5 at C-1, 3, 4, 6, 9, and
10 were same as 7 [21]. Because of all this evidence, the
chemical structure of valerianaterpene II (5) was determined
and shown in Fig. 1. This compound may have been isolated
as kessan 2-hydroxy-8-one, however, its chemical structure
especially absolute stereochemistry has not been described
in previous report [22, 23].
Valerianaterpene II (5) was isolated as a white amor-
phous powder with negative optical rotation ([α]25D -47.8
in MeOH). The molecular formula (C15H24O3) was deter-
1
mined using HRMS and 13C NMR spectroscopy. The H
and 13C NMR (CDCl3) spectra recorded for 5 (Table 1)
O
O
8
7
1
2
4
3
HO
1
5
15
HO
8
2
3
O
10
7
O
11
14
12
13
15
10
9
1
12
6
HO
5
11
13
14
Valerianaterpene III (10) was isolated as a white amor-
phous powder with positive optical rotation ([α]25D +17.8 in
MeOH). The molecular formula (C15H26O2) was determined
OH
10
using HRMS and 13C NMR spectroscopy. The 1H and 13
C
Fig. 2 Important 2D-NMR correlations of new constituents (1, 5, and
10)
NMR (CDCl3) spectra corresponding to three methyl groups
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Journal of Natural Medicines
[δH1.19 (s, H-12), 1.17 (s, H-13), and 1.21 (s, H-14)], two
quaternary carbons bearing oxygen function group [δC81.1
(C-3) and 74.2 (C-11)], and two olefnic carbons [δC153.3
(C-9) and 106.4 (C-15)] were similar but diferent to that
of pleocarpenene (3R,4R,6R,10R) [24] and diastereomer of
pleocarpenene (3R,4R,6R,10S) [25]. Therefore, the chemi-
cal structure of 10 was assumed to be diastereomer of above
described two compounds. The relative stereo structure of
10 was determined via NOESY spectra. Namely, cross-peaks
corresponding to H-1α/H-14, H-10/H-8α, and H-10/H-14
suggested that H-1α, H-8α, H-10, and H-14 were located on
same side. In addition, NOESY cross-peaks corresponding
to H-4/H-7 β, H-5 β/7 β, H-7 β/H-8 β, H-7 β/H-12, and H-7
β/H-13 suggested that H-4, H-5 β, H-7 β, H-8 β, H-11, H-12
and H-13 were located on same side. Therefore, the rela-
tive structure of 10 was determined as 3R*,4S*,6S*,10R*.
Finally, the absolute stereochemistry of 10 was deduced
as 3R,4S,6S,10R from the other isolated guaiane-type ses-
quiterpenes and positive optical rotation ([α]25D + 15.1 in
CHCl3) that was same tendency with that of 7 ([α]25D +6.7
in MeOH, recorded in this study) and known compound teu-
cladiol ([α]25D + 2.1 in CHCl3, data from literature) [26].
Because of all this evidence, the chemical structure of vale-
rianaterpene III (10) was determined and shown in Fig. 1.
ADR inhibits cell proliferation through induction of
G2/M cell cycle arrest. Previous study suggested that the
cell cycle arrest was induced via DNA damage; however,
low concentrations of ADR (0.1–1.0 µg/ml) does not induce
cell death [16]. Therefore, the compounds increase the num-
ber of dead cells on low concentration ADR treatment cells
may be able to reduce the dosage of ADR in tumor ther-
apy to avoid the side efects. We evaluated the cell death-
inducing activities of isolated compounds on HeLa cells by
24 h using time-lapse imaging analysis. In this study, we
counted the number of mitotic entry cells and dead cells
under treatment of low concentration ADR (1.0 µg/ml),
test compounds (1–11) (60 µM), and combination of test
compounds (60 µM) with ADR (1.0 µg/ml). As the results,
ADR signifcantly reduced the mitotic entry cells but did
not increase the dead cells same as previous report [27].
All isolated compounds (1–11, 60 µM) treatment did not
afect the number of the dead cells and the mitotic entry cells
(Fig. 4A). However, combination treatment of 7, 9, and 11
with ADR signifcantly increased the dead cells compared
to those of ADR treated cells (Fig. 4B). Above results sug-
gested that 7, 9, and 11 may have the potency for cancer
treatment agents without show side efects (Fig. 5).
Fig. 4 Efects of the isolated compounds (1–11) on cell prolifera-
tion and cell death. (A) HeLa cells were treated with 60 µM indicated
compounds, (B) ADR (1.0 µg/ml) or combination of them for 24 h.
The number of mitotic entry cells and dead cells was counted during
time-lapse imaging. The percentages of mitotic entry cells or dead
cells are reported as means SD of triplicate. Statistical signifcance
was analyzed using the Tukey–Kramer test (*P<0.05, **P<0.01,
***P<0.001 compared with ADR (1.0 µg/ml)-treated cells)
Fig. 5 The expression levels Hsp105, 90, and 70 on HeLa cell treated
with 9 for 24 h
that have benzylidene lactam structure was reported as
Hsp105 inhibitor [28, 29]. Hsp70 is also have anti-apop-
totic functions and it is induced by Hsp105 [30]. On the
other hand, inhibition of Hsp90 was reported to induce
other Hsps such as Hsp70 [31]; therefore, the compounds
inhibit Hsp105 and 70 without afect Hsp90 should be
new cancer treatment agents. The HeLa cell is the one of
the Hsp105 overexpression cancer cell line and siRNA-
mediated knockdown of Hsp105 enhances ADR-induced
cell death [16]. Among the isolated compounds in this
study, 9 showed strongest cell death-inducing activity on
ADR treatment cell; therefore, we evaluated expression
levels of Hsp105, 90, and 70 in 9 treated HeLa cell using
western blotting analysis. As the result, 9 inhibited the
expression of Hsp105 and 70 without afects the Hsp90
at 30 µm. The expression level of Hsp90 was weakly sup-
pressed by 60 µm treatment of 9. Based on above results,
we concluded that 9 is able to enhance the efects of pre-
vious cancer treatment agents such as ADR via inhibition
of Hsp105 expression. In the vest of our knowledge, this
Hsp105 is a molecular chaperone, and it was reported to
suppress ADR-induced cell death via anti-apoptotic func-
tions [16]. Because Hsp105 is overexpressed in several
kinds of tumor, inhibition of Hsp105 have been suggested
to be a target for cancer chemotherapy [26]. Previously,
only one small molecule organic compound, KNK437
1 3

Journal of Natural Medicines
is the frst report deals with the naturally occurring com-
(18.0 mg). From the fraction 4–3 (6785.7 mg), 9 (4.9 g) was
obtained by recrystallization (n-hexane–CHCl3). Fraction
4–4 (2759.2 mg) was purifed using HPLC {H2O–CH3CN
(40:60, v/v)} to give 1 (5.7 mg), 3 (61.1 mg), 4 (338.7 mg), 6
(6.1 mg), 8 (173.0 mg), and 11 (5.2 mg). Fraction 5 (2.98 g)
was separated using reversed-phase silica gel column chro-
matography to give fve fractions. Fraction 5–1 (477.8 mg)
was purified using HPLC {H2O–CH3CN–CH3COOH
(80:20:0.3, v/v/v)} to give 7 (98.2 mg). Fraction 5–3
(852.5 mg) was purifed using HPLC {H2O–CH3CN (65:35,
v/v)} to give 2 (8.9 mg) and 10 (5.8 mg).
pounds as Hsp105 inhibitor.
Experimental section
General experimental procedures
Specifc rotations were obtained on a JASCO P-2200 digital
polarimeter (l=5 cm). EIMS and HREIMS were recorded
1
on JEOL JMS-GCMATE mass spectrometer. H NMR
spectroscopy was recorded on JEOL ECS400 (400 MHz)
and JNM-ECA 600 (600 MHz) spectrometers. 13C NMR
spectroscopy was recorded on a JNM-ECA 600 (150 MHz)
spectrometer. 2D-NMR experiments were carried out on a
JEOL JNM-ECA 600 (600 MHz) spectrometer.
Valerianaterpene I (1)
Colorless needle crystals; [α]25D +8.8 (c 0.14, MeOH); 1H
NMR (CDCl3, 600 MHz) and 13C NMR (CDCl3, 150 MHz),
see Table 1; EIMS m/z 280 [M]+; HREIMS m/z 280.2036
[M]+ (calcd for C17H28O3, 280.2039).
Normal-phase silica gel column chromatography was
carried out using Silica gel 60 (Kanto Chemical Co., Inc.
63–210 mesh) and reversed-phase silica gel column chro-
matography was carried out on C18-OPN (Nakalai Tesque
Co., Inc. 140 µm). Thin-layer chromatography (TLC) was
performed using TLC plates pre-coated with 60F254 silica
gel (Merck, 0.25 mm; ordinary phase) and RP-18 F254S sil-
ica gel (Merck, 0.25 mm; reversed-phase). Reversed-phase
high-performance TLC was carried out using TLC plates
pre-coated with RP-18 WF254S silica gel (Merck, 0.25 mm).
HPLC was performed using a Shimadzu SPD-M10Avp
UV–vis detector. COSMOSIL 5C18-MS-II (250×4.6 mm
i.d., 250×10 mm i.d. and 250×20 mm i.d.) and YMC-Triart
PFP (250×4.6 mm i.d. and 250×10 mm i.d.) columns were
used for analytical and preparative purposes.
X‑ray difraction analysis of valerianaterpene I (1)
A colorless needle crystal of C17H28O3 with approximate
dimensions of 0.200× 0.100× 0.050 mm was mounted on
a Dual-Thickness MicroMouunt™. All measurements were
performed using a Rigaku R-AXIS RAPID II difractom-
eter with graphite monochromated Cu-Kα radiation. The
crystal-to-detector distance was 127.40 mm. MW 280.41,
monoclinic, space group P212121 (#19), a = 6.5826(3)
Å, b = 14.2197(7) Å, c = 17.2972(8) Å, V = 1619.07(13)
Å3, Z = 4, Dcalcd = 1.150 g/cm3, µ(Cu-Kα) = 6.087 cm–1,
F(000)=616.00, No. of refections measured: Total, 17,862;
unique, 2927 (Rint =0.0434); Flack parameter: 0.05(9). Fur-
ther crystallographic data regarding the compound structure
can be found in the supporting information. Crystallographic
data for 1 have been deposited with the Cambridge Crystal-
lographic Data Centre (deposition number CCDC 2084835).
Plant material
The rhizomes and roots of V. fauriei distributed in the Hok-
kaido of Japan were purchased from Tochimoto Tenkaido
(Osaka prefecture, Japan) in August 2020.
Valerianaterpene II (5)
Extraction and isolation
White amorphous powder; [α]25D -47.8 (c 0.37, MeOH); 1H
NMR (CDCl3, 600 MHz) and 13C NMR (CDCl3, 150 MHz),
see Table 1; EIMS m/z 252 [M]+; HREIMS m/z 252.1725
[M]+ (calcd for C15H24O3, 252.1726).
The rhizomes and roots of V. fauriei (6 kg) were extracted
three times with methanol under refux for 3 h. Evaporation
of the solvent provided a methanol extract (1150 g). The
methanol extract was partitioned into ethyl acetate–water
(1:1, v/v) to furnish an ethyl acetate-soluble fraction
(190.1 g, 3.1%) and water-soluble fraction (959.9 g, 8.0%).
The ethyl acetate-soluble fraction was subjected to normal-
phase silica gel column chromatography [n-hexane–CHCl3
(1:1→0:1, v/v)→CHCl3–MeOH (50:1→20:1→10:1→5
:1→1:1, v/v)] to give ten fractions. Fraction 4 (30.1 g) was
separated using reversed-phase silica gel column chromatog-
raphy to give nine fractions. Fraction 4–2 (1015.2 mg) was
purifed using HPLC {H2O–CH3CN (70:30, v/v)} to give 5
NaBH4 reduction of 5
To a solution of 5 (5.0 mg, 0.02 mmol) in MeOH (5 mL) was
added NaBH4 (5 mg, 0.13 mmol) and the mixture was stirred
for 0.5 h at 0 °C. Saturated aqueous NaHCO3 was added,
and the solution was extracted by CH2Cl2. The residue was
purifed using HPLC {H2O–CH3CN (65:35, v/v)} to give 7
(1.0 mg, 0.004 mmol, 19.8%) and 5a (0.6 mg, 0.0024 mmol,
11.9%).
1 3

Journal of Natural Medicines
treatment groups. The signifcance level used for statistical
Data for 7 derived from 5
analysis with two-tailed testing was 5%.
White amorphous powder; [α]25 -24.8 (c 0.05, MeOH);
D
1H NMR (CDCl3, 600 MHz) and 13C NMR (CDCl3,
150 MHz) were identical with literature [9]; EIMS m/z 254
[M]+; HREIMS m/z 254.1886 [M]+ (calcd for C15H26O3,
254.1882).
Western blotting analysis
HeLa cells (1.0×105 cells) were seeded into 35-mm dishes.
After 24 h of incubation, the cells were treated with vari-
ous concentrations of compounds for 24 h. Cells were lysed
with sodium dodecyl sulfate (SDS)‐sample bufer (2% SDS,
10% glycerol, 5% 2-mercaptoethanol, 62.5 mM Tris‐HCl
[pH 6.8], and 0.0125% bromophenol blue) and boiled at 100
℃ for 5 min. Proteins were separated by SDS-PAGE and
transferred onto polyvinylidene difuoride membranes (Pall
Corporation, Port Washington, NY). Blots were incubated
with Blocking One (Nacalai Tesque), and sequentially incu-
bated with appropriate primary and horseradish peroxidase
(HRP)-conjugated secondary antibodies that were diluted
with Tris-bufered saline containing 0.1% Tween 20 and 5%
Blocking One. Chemiluminescence was detected with an
image analyzer LAS-4000 mini system (Fujiflm, Tokyo,
Japan) using Clarity Western ECL Substrate (Bio‐Rad, Her-
cules, CA). Antibodies used in this study were as follows:
mouse monoclonal anti-Hsp105 (1:1000–2000; E5Q6W,
Santa Cruz Biotechnology, Dallas, TX, USA), anti-Hsp90
(1:2000; clone AC88, Enzo Life Sciences, Farming dale,
NY, USA), anti-Hsp70 (1:2000; clone C92F3A-5, Enzo Life
Sciences), and anti-α-tubulin (1:2000; clone DM1A, Sigma-
Aldrich, St. Louis, MO, USA) antibodies. HRP-conjugated
goat anti-mouse IgG (1:4000; 712–035-151) were purchased
form Jackson ImmunoResearch Laboratories Inc. (West
Grove, PA, USA).
Data for 5a derived from 5
White amorphous powder; [α]25 -31.7 (c 0.37, MeOH);
D
1H (CDCl3, 600 MHz) and 13C NMR (CDCl3, 150 MHz),
see Table 1; EIMS m/z 254 [M]+; HREIMS m/z 254.1882
[M]+ (calcd for C15H26O3, 254.1882).
Valerianaterpene III (10)
White amorphous powder; [α]25D + 17.8 (c 0.14, MeOH);
1H NMR (CDCl3, 600 MHz) and 13C NMR (CDCl3,
150 MHz), see Table 1; EIMS m/z 238 [M]+; HREIMS
m/z 238.1930 (calcd for C15H26O2 [M]+, 238.1933).
Cells
Human cervical carcinoma (HeLa) cells were maintained
in Dulbecco’s modifed Eagle’s medium (DMEM) with
low glucose (Wako Pure Chemical Industries, Osaka,
Japan) supplemented with 5% fetal bovine serum (Merck,
Darmstadt, Germany) under a 5% CO2 atmosphere at 37
℃.
Supporting information
1
The H and 13C NMR spectra for 1, 5, and 10 and crys-
Time‑lapse imaging
tallographic data for 1 are available in the supporting
Time-lapse imaging was performed on an Operetta high-
content imaging system (PerkinElmer, Waltham, MA) as
described previously [13]. Briefy, the cells were cultured
in a fat-bottomed 24-well plate (Coaster 3526; Corning) to
reach 70–80% confuence. The cells were then treated with
test compounds or Adriamycin just prior to the time-lapse
cell imaging. The images were captured at 10 min intervals
for 24 h under a 5% CO2 atmosphere at 37 ℃.
information.
Supplementary Information The online version contains supplemen-
tary material available at https://doi.org/10.1007/s11418-021-01543-9.
Acknowledgements This work was supported by JSPS KAKENHI
Grant Numbers 20H03397 and 20J14567.
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