Phenolic Constituents of the Roots of Rhamnoneuron balansae with Senolytic Activity

Article abstract of DOI:10.1021/acs.jnatprod.0c00885

With the advent of senolytic agents capable of selectively removing senescent cells in old tissues, the perception of age-associated diseases has been changing from being an inevitable to a preventable phenomenon of human life. In the search for materials with senolytic activity from natural products, six new flavonostilbenes (1-6), three new phenylethylchromanones (7-9), three new phenylethylchromones (10-12), and four known compounds (13-16) were isolated from the roots of Rhamnoneuron balansae. The chemical structures of these isolated compounds were determined based on the interpretation of spectroscopic data, including 1D and 2D NMR, ECD, and HRMS. The absolute configuration of compound 1 was also determined by a Mosher ester analysis and ECD calculations. Compounds 6-8 were shown to selectively destroy senescent cells, and the promoter activity of p16INK4A, a representative senescence marker, was reduced significantly by compound 6. The present results suggest the potential activity of flavonostilbene and phenylethylchromanone skeletons from R. balansae as new senolytics.

Full text of DOI:10.1021/acs.jnatprod.0c00885

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Article
Phenolic Constituents of the Roots of Rhamnoneuron balansae with
Senolytic Activity
Hyo-Moon Cho, Yae-Rin Lee, Ba-Wool Lee, Mi Zhang, Byeol Ryu, Du-Trong Nghiem,
Ha-Thanh-Tung Pham, and Won-Keun Oh*
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ABSTRACT: With the advent of senolytic agents capable of
selectively removing senescent cells in old tissues, the perception of
age-associated diseases has been changing from being an inevitable
to a preventable phenomenon of human life. In the search for
materials with senolytic activity from natural products, six new
flavonostilbenes (1−6), three new phenylethylchromanones (7−
9), three new phenylethylchromones (10−12), and four known
compounds (13−16) were isolated from the roots of Rhamnoneur-
on balansae. The chemical structures of these isolated compounds
were determined based on the interpretation of spectroscopic data,
including 1D and 2D NMR, ECD, and HRMS. The absolute
configuration of compound 1 was also determined by a Mosher
ester analysis and ECD calculations. Compounds 6−8 were shown to selectively destroy senescent cells, and the promoter activity of
p16INK4A, a representative senescence marker, was reduced significantly by compound 6. The present results suggest the potential
activity of flavonostilbene and phenylethylchromanone skeletons from R. balansae as new senolytics.
he average life expectancy for people born in the U.S. in
1959 has risen from 69.9 years to 78.9 years,1 which is
ultimately resulting in tissue damage and immune system
disturbance.6 To overcome this chronic senescence, anti-
senescent drugs have been presented as “senolytics” that
specifically eliminate senescent cells by inducing apoptosis and
“senomorphics” that can suppress senescence indirectly by
SASP inhibition.7 During cell cycle arrest, the p53/p21WAF1
axis and p16INK4A play their roles in the initiation of
senescence and the maintenance of state, respectively.8
Proteins of the B-cell lymphoma 2 (BCL-2) family, ephrin-
dependent receptors, HSP90, PI3Kδ/Akt, and FOXO4a/p21,
which comprise the senescent cell antiapoptotic pathways
(SCAPs), have been suggested to be involved in apoptosis
resistance.9 As SASP inhibitors, many studies on the related
pathways of NF-κB, mTOR, and p38MAPK have been
proposed.10 Dasatinib (an inhibitor of the Eph receptors),
quercetin (a modulator of several pathways, such as NF-κB and
mTOR), navitoclax (an inhibitor of the BCL-2 family
proteins), and fisetin are reported to have senolytic
activity.11−14 Apigenin and kaempferol, rapamycin (a specific
inhibitor of mTOR), and ruxolitinib (a JAK inhibitor) have
T
attributed to improved medical care, knowledge of hygiene,
and access to vaccinations. The U.S. and other wealthy
countries have kept pace with this tendency over the past few
decades. However, since a prolonged life span is not always
proportional to an individual’s health, interest has grown as to
how to age well by preserving physical and mental health to
maximize the quality of life during one’s elderly years.2 Aging
accompanies the progressive accumulation of structural and
functional damage in an organism and is prone to result in
multiple and serious chronic diseases, including Alzheimer’s
disease, osteoporosis, diabetes, frailty, and lung diseases.3
Thus, many researchers have investigated fundamental aging
mechanisms, which include cellular senescence.
Cellular senescence is a state in which cells cannot
permanently replicate but are viable metabolically and
triggered by various stimuli, including DNA damage (e.g.,
telomere shortening), reactive oxygen species (ROS) gen-
eration, and oncogenes (e.g., Ras and Myc).4 Senescent cells
are characterized by particular morphological and metabolic
changes, including enlarged cell shapes and resistance against
apoptosis, chromatin reorganization, altered gene expression,
and the acquisition of the senescence-associated secretory
phenotype (SASP), including proinflammatory cytokines,
bradykinins, chemokines, proteases, and miRNAs.5 Senescent
cells accumulate in various tissues, and the secretion of SASP
by these aged cells causes chronic inflammation in normal cells,
Received: August 8, 2020
© XXXX American Chemical Society and
American Society of Pharmacognosy
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been suggested to be interesting senomorphics.15−18 Recently,
promising results have been reported in several clinical studies
where a combined treatment of dasatinib and quercetin
significantly reduced SASP factors and the burden of senescent
cells. Thus, research is starting to find senotherapeutics from
natural resources with favorable safety profiles.19
Rhamnoneuron balansae (Drake) Gilg is a medium-sized tree
belonging to the family Thymelaeaceae and is distributed
throughout northern Vietnam and mainland China. The plant
at δH 6.10 (2H, d, J = 2.0 Hz) and 6.15 (1H, d, J = 2.1 Hz),
two methines of a dihydroflavonol moiety at δH 5.02 and 4.47
(each 1H, d, J = 11.6 Hz), and two methines of a
dihydrobenzofuran moiety at δH 5.52 and 4.29 (each 1H, d,
J = 5.1 Hz). The 13C NMR data (Table 3) showed all 29
expected carbon resonances. Fifteen of the carbon signals were
explained by a dihydrokaempferol skeleton, including four
oxygenated aromatic carbons and one carbonyl carbon at δC
159.1, 163.0, 163.5, 165.2, and 192.9. The remaining 14
carbon signals suggested the presence of the fused stilbene
moiety (C6−C2−C6) by heteronuclear multiple bond
correlations (HMBC) (Figure 1). The HMBC correlations
between H-2′/6′ (δH 7.38, 6.84) and C-2 (δC 84.9) and of H-8
(δH 5.98) and C-6 (δC 111.1), C-7 (δC 163.0), C-9 (δC 165.2),
and C-10 (δC 100.8) showed the presence of dihydroflavonol
with a 1,4-disubstituted B-ring. A 1,2-diaryl-dihydrobenzofuran
ring containing 1,4-disubstituted and 1,3,5-trisubstituted
benzene rings was suggested by the HMBC correlations from
H-7″ (δH 5.52) to C-1″ (δC 133.3) and C-2″/6″ (δC 128.2), as
well as from H-8″ (δH 4.29) to C-9″ (δC 146.2) and C-10″/
14″ (δC 106.7). The linkage of the resveratrol and
dihydrokaempferol moieties was confirmed by the HMBC
correlations from H-7″ to C-5 (δC 163.5) and C-6 and from H-
8″ to C-5, C-6, and C-7. The relative configuration of
compound 1 were assigned all-trans orientations by the nuclear
Overhauser effect spectroscopy (NOESY) correlations (Figure
2A and B) between H-3 and H-2′/6′, H-7″ and H-10″/14″,
and H-8″ and H-2″/6″, respectively. These assignments were
supported by the large J2,3 value of 11.6 Hz and the small J7″,8″
value of 5.1 Hz in comparison to those from previous
studies.21−23 The absolute configurations of C-2 and C-3 were
determined as 2S,3S based on Mosher ester analysis (Figures
S94−97 and Table S1, Supporting Information).24 Moreover,
the remaining absolute configuration of the dihydrobenzofuran
moiety was assigned by a comparative analysis of the
experimental electronic circular dichroism (ECD) spectra
using the time-dependent density functional theory
(TDDFT) method at the 6-31G/B3LYP level (Figure 3A).
By considering the molecular orbital transitions and comparing
the experimental data with the calculated data, the absolute
configuration of 1 was suggested as 2S,3S based on a negative n
→ π* Cotton effect (CE) of 300−340 nm and 7″S,8″S based
on a negative CE of 240 nm (Figure S101 and Table S24,
Supporting Information). From all of the aforementioned
observations, the absolute configuration of compound 1 was
defined as 2S,3S,7″S,8″S, and this compound was named
rhamnoneuronal A.
́
has been utilized as a raw material for Do paper, also known as
bamboo paper, and has been used in traditional Vietnamese
́
crafts for centuries. Do paper exhibits no smudging of ink, is
resistant to moisture and acid, and offers protection from
termites. The durability of this paper makes it an important
source for folk crafts. Despite being used for centuries, little has
been reported about its chemical constituents, including
studies on its pharmacological activity.20 Herein are reported
the isolation and structure determination of 12 new
compounds from a 70% ethanol extract of the roots of R.
balansae. In addition, the pharmacological activity of R.
balansae and its constituents was investigated, and rhamno-
neuronal C (6) and balansechromanones A (7) and B (8)
were found to be potential senolytic candidates.
RESULTS AND DISCUSSION
■
A 70% ethanol extract of the dried roots of R. balansae was
applied to a series of chromatographic techniques, including
passage over silica gel, reversed-phase-C18, and Sephadex LH-
20. Subsequently, preparative high-performance liquid chro-
matography (HPLC) was applied to obtain six flavonostilbenes
(1−6), three phenylethylchromanones (7−9), and three
phenylethylchromones (10−12) along with four known
compounds (13−16).
Compound 2 (rhamnoneuronal B) was isolated as a pale
yellow, amorphous powder with [α]2D5 −52 (c 0.1, MeOH).
The molecular formula, C29H22O10, was established from the
[M − H]− HRESIMS ion at m/z 529.1117 (calcd for
C29H21O10, 529.1140). The IR spectrum exhibited absorption
bands of hydroxy (3357 cm−1) and carbonyl (1612 cm−1)
Compound 1, obtained as a light yellow, amorphous powder
with [α]2D5 −15 (c 0.1, MeOH), was found to possess a
molecular formula of C29H22O9, based on the high-resolution
electrospray ionization mass spectrometry (HRESIMS) ion
peak at m/z 513.1192 [M − H]− (calcd for C29H21O9,
513.1191). The IR data exhibited the absorption of hydroxy
(3375 cm−1) and carbonyl (1659 cm−1) functionalities. The
1H NMR spectrum (Table 1) displayed signals for one
aromatic methine proton at δH 5.98 (1H, s), four sets of
protons from two 1,4-disubstituted benzene moieties at δH
7.38, 7.19, 6.84, and 6.79 (each 2H, d, J = 8.6 Hz), three
aromatic protons from one 1,3,5-trisubstituted benzene moiety
1
functionalities. The H and 13C NMR data (Tables 1 and 3)
displayed features similar to those of 1, except for the B-ring of
the dihydroflavonol unit. The 1D NMR spectra of 2 showed
that the B-ring of the dihydrokaempferol of 1 was replaced
with a dihydroquercetin unit from the correlations between H-
2′ at δH 6.99 (1H, d, J = 2.0 Hz; δC 115.9), H-5′ at δH 6.81
(1H, d, J = 8.1 Hz; δC 116.1), and H-6′ at δH 6.87 (1H, dd, J =
8.1, 2.0 Hz; δC 120.9). The absolute configuration of 2 was
defined as 2S,3S,7″S,8″S through comparative analysis with the
spectroscopic data of compound 1, including the coupling
B
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Table 1. NMR Spectroscopic H NMR Data for Compounds 1−6
b
b
b
b
b
b
1
2
3
4
5
6
position
δH (J in Hz)
δH (J in Hz)
δH (J in Hz)
δH (J in Hz)
δH (J in Hz)
δH (J in Hz)
2
3
8
5.02, d (11.6)
4.47, d (11.6)
5.98, s
4.95, d (11.5)
4.42, d (11.5)
5.98, s
5.02, d (11.6)
4.48, d (11.6)
5.98 s
5.07, d (11.7)
4.53, d (11.7)
6.35, s
5.06, d (11.8)
4.57, d (11.8)
6.35, s
5.10, d (11.9)
4.60, d (11.9)
6.20, s
2′
3′
7.38, d (8.6)
6.84, d (8.6)
6.99, d (2.0)
7.38, d (8.5)
6.85, d (8.5)
7.38, d (8.5)
6.84, d (8.5)
7.39, d (8.5)
6.84, d (8.5)
7.41, d (8.5)
6.86, d (8.5)
4′
5′
6′
6.84, d (8.6)
7.38, d (8.6)
6.81, d (8.1)
6.87, dd (8.1, 2.0)
6.85, d (8.5)
7.38, d (8.5)
6.84, d (8.5)
7.38, d (8.5)
6.84, d (8.5)
7.39, d (8.5)
6.86, d (8.5)
7.41, d (8.5)
1″
2″
3″
4″
5″
6″
7″
8″
9″
10″
11″
12″
13″
14″
Glc
1‴
2‴
3‴
4‴
5‴
6‴
7.19, d (8.6)
6.79, d (8.6)
7.18, d (8.5)
6.79, d (8.5)
7.31, d (8.7)
7.13, d (8.7)
7.18, d (8.5)
6.79, d (8.5)
7.14, d (8.6)
6.78, d (8.6)
7.45, d (8.8)
6.71, d (8.8)
6.79, d (8.6)
7.19, d (8.6)
5.52, d (5.1)
4.29, d (5.1)
6.79, d (8.5)
7.18, d (8.5)
5.52, d (5.2)
4.28, d (5.2)
7.13, d (8.7)
7.31, d (8.7)
5.58, d (5.0)
4.27, d (5.0)
6.79, d (8.5)
7.18, d (8.5)
5.62, d (5.6)
4.33, d (5.6)
6.78, d (8.6)
7.14, d (8.6)
5.65, d (5.2)
4.32, d (5.2)
6.71, d (8.8)
7.45, d (8.8)
6.10, d (2.0)
6.15, t (2.1)
6.10, d (2.0)
6.10, d (2.2)
6.15, t (1.6)
6.10, d (2.2)
6.11, d (2.1)
6.16, t (2.2)
6.11, d (2.1)
4.93, d (7.5)
6.14, d (2.1)
6.17, t (2.1)
6.14, d (2.1)
6.15, d (2.1)
6.16, t (2.1)
6.15, d (2.1)
6.38, d (2.2)
6.30, t (1.8)
6.38, d (2.2)
d
d
4.85, d
4.88, d
d
3.46, m
3.44, m
3.39, m
3.46, m
3.20, dd (9.0, 7.9)
3.34, t (9.1)
3.30, t (9.2)
3.37, m
3.81, dd (12.1, 2.0)
3.22, dd (9.0, 7.9)
3.30, t (9.1)
3.00, t (9.2)
3.35, m
3.83, dd (12.1, 2.1)
d
d
d
f
3.91, dd (12.1, 5.7)
3.72 dd (12.1, 2.2)
3.62, dd (12.1, 5.5)
3.64, dd (12.1, 5.3)
a
b
c
d
Recorded in methanol-d4 at 400 MHz. Recorded in methanol-d4 at 800 MHz. Recorded in CDCl3 at 800 MHz. Overlapped.
constants, a NOESY experiment, and the ECD spectrum
(Figure 3B). Accordingly, compound 2 was assigned as shown.
Compound 3 (rhamnoneuroside A) was obtained as a light
yellow, amorphous powder with [α]2D5 −98 (c 0.1, MeOH).
The molecular formula, C35H32O14, was established from the
deprotonated HRESIMS ion peak at m/z 675.1716 (calcd for
C35H31O14, 675.1714). The IR spectrum exhibited absorption
bands for hydroxy (3389 cm−1) and carbonyl (1600 cm−1)
functionalities. The NMR data of compound 3 were very
similar to those of 2 except for the presence of a β-D-glucose
moiety. The β-glucopyranosyl unit was deduced through a
large coupling constant of the anomeric proton at δH 4.93 (1H,
d, J = 7.5 Hz; δC 102.2), and the connectivity of six protons
functionalities. A comparison of the 1D and 2D NMR data of 1
with 4 showed that the additional signals of an anomeric
proton at δH 4.85 (1H, d), corresponding to the carbon
resonance at δC 101.3, indicated the presence of a β-D-glucose
moiety. The HMBC correlation between H-1‴ of glucose and
C-7 suggested that the glucose residue is located at C-7 of a
dihydrokaempferol moiety. The absolute configuration of
compound 4 (2S,3S,7″S,8″S) was defined through the close
similarity of the ECD curve with that of compound 1.
Consequently, compound 4 was assigned as shown.
Compound 5 (rhamnoneuroside C) was obtained as a light
yellow, amorphous powder with [α]2D5 −45 (c 0.1, MeOH).
The molecular formula of 5 was determined as C35H32O14
from the [M − H]− HRESIMS ion peak at m/z 675.1716
(calcd for C35H31O14 675.1714). The IR spectrum exhibited
absorption bands for hydroxy (3361 cm−1) and carbonyl (1617
cm−1) groups. The NMR spectra of 5 were very similar to
those of 4, whereas the respective ECD analysis of compounds
4 and 5 indicated them to be diastereoisomers. The key
rotating-frame Overhauser enhancement spectroscopy
(ROESY) correlations (Figure 2A and C) between H-3 and
H-2′/6′ and the large coupling constant (J2,3) showed that the
relative configuration of H-2 and H-3 of 5 is the same as in
compound 4 with a trans conformation. However, the positive
n → π* CE at 300−340 nm of compound 5 suggested that the
absolute configuration of the dihydrokamferol unit appeared
different from that of 4 (Figure 3C).25 Therefore, the absolute
1
appearing in the aliphatic region was revealed by the H−1H
COSY experiment. The HMBC correlation between H-1‴ (δH
4.93) and C-4″ (δC 159.1) suggested that the glucose group in
compound 3 is attached to C-4″ of the stilbene moiety. The
absolute configuration of compound 3 (2S,3S,7″S,8″S) was
assigned through a comparative ECD analysis (Figure 3B).
Accordingly, the structure of compound 3 was determined as
shown.
Compound 4 (rhamnoneuroside B) was isolated as a light
yellow, amorphous powder with [α]2D5 −140 (c 0.1, MeOH).
The molecular formula, C35H32O14, was established from the
[M − H]− HRESIMS ion peak at m/z 675.1724 (calcd for
C35H31O14, 675.1719). The IR spectrum exhibited absorption
bands for hydroxy (3347 cm−1) and carbonyl (1616 cm−1)
C
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Table 2. NMR Spectroscopic H NMR Data for Compounds 7−10
a
a
a
b
c
c
7
8
9
10
11
12
position
δH (J in Hz)
δH (J in Hz)
δH (J in Hz)
δH (J in Hz)
δH (J in Hz)
δH (J in Hz)
2
3
4.43, m
4.42, m
4.37, m
2.62, dd (16.8, 3.1)
2.72, m
2.67, dd (16.8, 2.8)
2.67, dd (16.8, 3.0)
d
d
2.74, m
2.74, m
4
5
6
7
8
7.81, d (7.8)
7.02, t (7.9)
7.54, t (8.7)
7.05, d (8.5)
7.81, d (7.8)
7.02, t (7.9)
7.54, t (8.7)
7.05, d (8.3)
7.78, d (7.5)
8.22, d (7.9)
7.37, t (7.4)
7.64, t (8.5)
7.46, d (8.4)
8.22, d (7.9)
7.37, t (7.5)
7.64, t (7.7)
7.44, d (8.4)
8.22, d (7.9)
7.37, t (7.5)
7.64, t (7.7)
7.45, d (8.4)
d
6.98
7.49, d (7.3)
7.00e
9
10
1′
2′
3′
4′
5′
d
7.06, d (8.5)
6.70, d (8.5)
6.81, d (1.8)
6.68
6.73, br s
6.67, br s
6.75, br s
6.61, d (8.3)
7.06, t (7.7)
6.71, dd (8.0, 1.6)
7.14, t (7.8)
6.70, d (8.5)
7.06, d (8.5)
6.71, d (8.0)
6.82, d (8.5)
6.75, br d (8.5)
3.02, t (7.5)
6.82, d (8.0)
6.70, br d (8.0)
3.00, t (7.6)
3.08, t (7.6)
3.73, s
d
6′
7′
8′
6.67, dd (8.0, 1.9)
6.67
6.77, br d (7.9)
3.02, t (7.7)
3.11, t (7.7)
3.72, s
d
d
d
2.73, m
2.74, m
2.70, m
2.81, m
1.96, m
2.13, m
2.82, m
1.98, m
2.14. m
2.79, m
1.93, m
2.10, m
3.14, t (7.5)
MeO-3
MeO-3′
3.81, s
3.80, s
3.77, s
d
a
b
c
Recorded in methanol-d4 at 800 MHz. Recorded in CDCl3 at 400 MHz. Recorded in CDCl3 at 800 MHz. Overlapped.
configuration of the dihydrokaempferol moiety in the structure
of 5 was determined as 2R,3R based on the comparison of
ECD data. Thus, compound 5 was defined as 2R,3R,7″S,8″S
and the structure was assigned as shown.
through cyclization to produce compound 1. Thus, the
determined structure of compound 1 can be supported by
the cationic form (II), which is the most stable structure.
Finally, compound 1 can produce derivatives 3−5, in which
glucose is attached to the C-7 or C-4″ by O-glycosyl
transferase, and compound 6 can be suggested to occur, as
two methines in compound 1 are changed to olefins by
dehydrogenase.
Compound 6 (rhamnoneuronal C) was purified as a light
yellow, amorphous powder with [α]2D5 −10 (c 0.1, MeOH).
The molecular formula was determined as C29H20O9 from the
[M − H]− HRESIMS ion peak at m/z 511.1028 (calcd for
C29H19O9 511.1035). The IR spectrum exhibited absorption
bands for hydroxy (3336 cm−1) and carbonyl (1611 cm−1)
functionalities. By comparing the 1D and 2D NMR data of 6
with those of 1, compound 6 revealed a similar structure
except for the double bond at C-7″ and C-8″. Two mutually
coupled methines of 1 were replaced by the olefinic bond at C-
7″ (δC 151.7) and C-8″ (δC 116.1) in compound 6. This
assignment was further confirmed by its HMBC correlations
from H-2″/6″ at δH 7.45 (2H, d, J = 8.8 Hz) to C-7″ and from
H-10″/14″ at δH 6.38 (2H, d, J = 2.2 Hz) to C-8″. The
absolute configuration of compound 6 was determined to be
2S,3S compared to the ECD spectrum of 1. Accordingly,
compound 6 was assigned structurally as shown.
Compounds 1−6 have been proposed as a biosynthetic
pathway for new flavonostilbenes produced by the fusion of
resveratrol and dihydroflavonol in R. balansae (Scheme S1,
Supporting Information). Peroxidase uses two resveratrol units
to effectively oxidize and transfer electrons in phenols to
produce resveratrol-derived radicals (I).26 After the radical
form (I) is transformed into the cationic form (II) through
dismutase, the C−C bond was generated between resveratrol
and dihydroflavonol units by nucleophilic attack from the
resorcinol moiety in the A-ring of dihydroflavonol to the cation
of resveratrol.27 The highly reactive p-quinone methide is
attacked by hydroxy at C-6 to form a dihydrobenzofuran ring
Compound 7 (balansechromanone A) was isolated as a pale
yellow oil with [α]2D5 +83 (c 0.1, MeOH). A molecular formula
of C17H16O3 was established from the negative-ion HRESIMS
data at m/z 267.1015 [M − H]− (calcd for C17H15O3
267.1021). The IR spectrum exhibited absorption bands for
hydroxy (3361 cm−1) and carbonyl (1607 cm−1) groups. The
1H NMR data (Table 1) displayed signals for 1,2-disubstituted
benzene protons at δH 7.81 (1H, d, J = 7.8 Hz), 7.02 (1H, t, J =
7.9 Hz), 7.54 (1H, t, J = 8.7 Hz), and 7.05 (1H, d, J = 8.5 Hz),
1,4-disubstituted benzene protons at δH 7.06 (2H, d, J = 8.5
Hz) and 6.70 (2H, d, J = 8.5 Hz), two methylene protons at δH
2.81, 2.73, 2.13, and 1.96 (1H each, m), a set of mutually
coupled oxymethine proton at δH 4.43 (1H, m), and
methylene protons at δH 2.74 (1H, dd, J = 16.8, 2.8 Hz)
and 2.67 (1H, m). The 13C NMR data (Table 3) showed a
total of 17 carbon resonances, including two oxygenated
aromatic carbons at δC 156.7 and 163.3 and one carbonyl
carbon at δC 194.7. The presence of a 2-(2-phenylethyl)-
chromanone unit in compound 7 was determined based on the
HMBC correlations from H-2 to C-8′ along with those from
H2-7′ to C-1′ and C-2′/6′ (Figure 1). The absolute
configuration of compound 7 was assigned by means of an
ECD comparative analysis using TDDFT at the def-SV(P)/
B3LYP level (Figure 3D). The positive CE at 213 nm and the
negative CE at 296 nm with the chromanone moiety suggested
D
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Table 3. NMR Spectroscopic 13C NMR Data for Compounds 1−12
a
b
b
b
b
b
b
b
b
c
d
d
1
2
3
4
5
6
7
8
9
10
11
12
position
δC
δC
δC
δC
δC
δC
position
δC
δC
δC
δC
δC
δC
2
3
4
5
6
7
8
9
10
1′
2′
3′
4′
84.9
74.6
85.1
74.6
85.0
74.6
85.3
74.7
85.2
74.5
85.8
74.5
2
3
4
5
6
7
8
9
10
1′
2′
3′
4′
78.5
43.8
78.5
43.8
78.4
43.7
151.7
138.5
172.7
125.6
124.5
133.2
118.2
155.7
121.1
132.3
111.0
146.6
144.2
161.6
141.4
174.1
126.0
124.7
133.3
117.8
155.4
124.5
132.2
111.0
146.6
144.3
161.7
141.4
174.3
126.0
124.8
133.4
117.9
155.0
124.5
142.2
115.5
156.2
113.7
192.9
163.5
111.1
163.0
97.5
165.2
100.8
129.5
130.4
116.1
159.1
192.7
163.5
111.1
163.1
97.6
165.1
100.8
130.1
115.9
146.3
147.1
192.8
163.4
111.0
163.1
97.6
165.2
100.8
129.5
130.3
116.1
159.2
193.2
162.6
113.3
161.2
97.2
165.4
102.5
129.3
130.4
116.1
159.2
193.4
162.5
113.4
161.2
97.2
165.4
102.5
129.2
130.5
116.1
159.3
192.3
162.6
115.5
161.0
98.6
163.5
101.2
129.5
130.4
116.1
159.2
194.7
127.7
122.3
137.4
119.1
163.3
122.2
133.3
130.4
116.2
156.7
194.7
127.7
122.3
137.4
119.1
163.2
122.2
134.1
113.2
148.9
145.8
194.6
127.6
122.3
137.3
119.1
163.1
122.1
144.1
116.3
158.5
114.0
5′
6′
1″
2″
3″
4″
5″
6″
116.1
130.4
133.3
128.2
116.4
158.8
116.4
128.2
96.9
116.1
120.9
133.5
128.2
116.4
158.8
116.4
128.2
96.9
116.1
130.3
136.6
127.8
117.9
159.1
117.9
127.8
96.3
116.1
130.4
132.9
128.2
116.4
158.9
116.4
128.2
97.0
116.1
130.5
133.1
128.1
116.4
158.9
116.4
128.1
96.9
116.1
130.4
123.2
129.1
116.2
158.7
116.2
129.1
151.7
116.1
136.1
109.7
159.3
102.9
159.3
109.7
5′
6′
7′
8′
116.2
130.4
31.3
116.2
121.9
31.7
38.0
56.3
130.5
120.7
32.0
114.5
121.1
32.4
114.5
121.1
32.9
31.3
60.7
55.9
129.9
120.8
33.0
31.0
60.7
38.0
37.5
31.1
MeO-3
MeO-3′
56.0
7″
8″
9″
55.1
55.1
55.3
55.4
55.2
146.2
106.7
159.8
102.1
159.8
106.7
146.2
106.8
159.8
102.2
159.8
106.8
146.2
106.8
159.9
102.2
159.9
106.8
146.3
106.7
160.1
102.5
160.1
106.7
146.5
106.7
160.2
102.5
160.2
106.7
10″
11″
12″
13″
14″
Glc
1‴
2‴
3‴
4‴
5‴
6‴
102.2
74.9
78.0
71.4
78.2
62.5
101.4
74.7
77.2
70.9
78.3
101.3
74.7
77.4
70.9
78.3
62.2
62.2
b
a
c
d
Recorded in methanol-d4 and at 100 MHz. Recorded in methanol-d4 and at 200 MHz. Recorded in CDCl3 and at 100 MHz. Recorded in
CDCl3 at 200 MHz.
that the absolute configuration at C-2 is 2R. Therefore,
compound 7 was assigned as shown.
compound 7 with those of 8 indicated that the absolute
configuration of 8 is 2R (Figure 3D). Thus, compound 8 was
assigned as shown.
Compound 8 (balansechromanone B) was obtained as a
pale yellow oil with [α]2D5 +101 (c 0.1, MeOH). The molecular
formula was determined as C18H18O4 from the positive
HRESIMS ion at m/z 299.1281 [M + H]+ (calcd for
C18H19O4 299.1283). The IR spectrum exhibited absorption
signals for hydroxy (3426 cm−1) and carbonyl (1606 cm−1)
functionalities. Its NMR data indicated that compound 8 is
structurally similar to 7 except for the benzene system at the
phenylethyl moiety. The A2B2 system of the 2-phenylethyl in 7
was changed to an ABX system at δH 6.81 (1H, d, J = 1.8 Hz),
6.71 (1H, d, J = 8.0 Hz), and 6.67 (1H, dd, J = 8.0, 1.9 Hz). In
addition, compound 8 showed the presence of a methoxy
resonance at δH 3.81 (3H, s; δC 56.3). The position of the
methoxy group was located at C-3′ based on the HMBC cross-
peaks from the methoxy group to C-3′ (δC 148.9) and the
NOESY correlation between the methoxy group and H-2′ (δH
6.73). A comparison of the experimental ECD spectra of
Compound 9 (balansechromanone C) was obtained as a
pale yellow oil with [α]2D5 +85 (c 0.1, MeOH). The molecular
formula was determined as C17H16O3 from the negative
HRESIMS ion at m/z 267.1012 [M − H]− (calcd for
C17H15O3 267.1021). The IR spectrum exhibited absorption
bands for hydroxy (3362 cm−1) and carbonyl (1605 cm−1)
groups. The 1D and 2D NMR data were similar to 7 except for
the 1,3-disubstituted benzene group in compound 9. Its
HMBC correlations from H2-7′ at δH 2.79 and 2.70 to C-1′ (δC
144.1), C-2′ (δC 116.3), and C-6′ (δC 121.9) supported these
inferences. The absolute configuration of C-2 was determined
as the same as compound 7 based on its ECD spectrum
(Figure 3D). Thus, compound 9 was assigned as shown.
Compound 10 (balansechromone A) was isolated as a pale
yellow oil with [α]2D5 +60 (c 0.1, MeOH). The molecular
formula of this compound was determined as C18H16O5 from
E
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(2H, t, J = 7.5 Hz) and 3.02 (2H, t, J = 7.5 Hz), and a methoxy
group at δH 3.80 (3H, s). The 13C NMR spectrum (Table 3) of
10 showed a set of olefinic carbons at δC 151.7 and 138.5 and a
conjugated carbonyl carbon at δC 172.7. The presence of the
methoxy group at C-3 was deduced by the HMBC correlation
from MeO-3′ at δH 3.80 to C-3′ (δC 146.6) and the NOESY
correlation between MeO-3′ and H-2′ (δH 6.73). The
comparison of the 1D and 2D NMR data of compound 10
with those of 8 suggested that H-2 (δC 78.5) and H2-3 (δC
43.8) in compound 8 were replaced by a 2,3-olefinic bond at
δC 151.7 and 138.5 in 9. Its HMBC correlations from H2-8′ to
C-2 and C-3 confirmed that compound 9 has a 3-
hydroxychromone moiety (Figure 1). Thus, compound 10
was determined to be a 3-hydroxy-2-(2-phenylethyl)chromone
derivative as shown.
Compound 11 (balansechromone B) was isolated as a light
yellowish oil with [α]2D5 +64 (c 0.1, MeOH). Its molecular
formula was determined as C19H18O5 from the [M + H]+
HRESIMS ion at m/z 327.1232 (calcd for C19H19O5
327.1232). The IR spectrum exhibited absorption signals for
hydroxy (3362 cm−1) and carbonyl (1618 cm−1) function-
alities. A comparative analysis of the NMR spectra of
compounds 10 and 11 showed the presence of an additional
methoxy group at δH 3.73 (3H, s; δC 60.7) in 11. The position
of the additional methoxy group was suggested by the HMBC
correlation between MeO (δH 3.73) and C-3 (δC 141.4).
These structural changes were confirmed by the downfield shift
of C-2 and C-3 from δC 151.7 and 138.5 of 10 to δC 161.6 and
141.4 of compound 11. Finally, compound 11 was assigned as
shown.
Compound 12 (balansechromone C) was isolated as a light
yellowish oil with [α]2D5 +63 (c 0.1, MeOH). The molecular
formula of 12 was determined to be C18H16O4 from its [M −
H]− HRESIMS ion peak at m/z 295.0977 (calcd for C18H15O4
295.0970). The IR spectrum exhibited the absorption signals
of hydroxy (3331 cm−1) and carbonyl (1617 cm−1)
functionalities. On comparing the NMR spectra of compound
12 with those of 11, the structure of 12 was similar to 11
except for the signals for the benzene ring of the 2-phenylethyl
moiety. The 1,3,4-trisubstituted benzene moiety in 11 was
replaced by a 1,3-disubstituted benzene group in 12, and this
assignment was deduced by the proton and carbon signals at
δH 7.14 (1H, t, J = 7.8 Hz; δC 129.9), 6.77 (1H, br d, J = 7.9
Hz; δC 120.8), 6.75 (1H, br s; δC 115.5), and 6.71 (1H, dd, J =
8.0, 1.6 Hz; δC 113.7). These results were confirmed through
the HMBC correlations from H2-7′ (δH 3.02) to C-1 (δC
142.2), C-2 (δC 115.5), and C-6 (δC 120.8).28 Therefore,
compound 11 was assigned as shown.
1
Figure 1. Key HMBC (red arrows) and H−1H COSY (bold black
lines) correlations of compounds 1−12.
Based on the NMR data and optical rotations as well as
literature values, four known compounds were elucidated as
(2R,4aS,7R,8aR)-7-(3-hydroxyprop-1-en-2-yl)-4a-methyl-1-
methylenedecahydronaphthalen-2-ol (13),29 viscic acid (14),30
(+)-hinokinin (15),31 and (2R,3S)-2-hydroxy-2,3-bis(4-hy-
droxy-3-methoxybenzyl)-4-butanolide (16).32
To explore the senolytic activity of the new isolated
compounds, the ability to selectively kill senescent cells was
evaluated by exposing the compounds to proliferating cells and
senescent human dermal fibroblast (HDF) cells and comparing
the results. The 12 new compounds isolated from R. balansae,
at a 20 μM concentration, were used to treat proliferating cells,
and toxicity was not observed (Figure 4A). Based on this, cell
viability was evaluated by treating senescent cells with the
isolated compounds at the same concentration. Among these
Figure 2. Key ROESY/NOESY correlations of compounds 1−5 (A).
3D model of the lowest energy conformation of compounds 1 (B)
and 5 (C).
the negative HRESIMS ion at m/z 311.0913 [M − H]− (calcd
for C18H15O5 311.0919). The IR absorption bands showed the
presence of hydroxy (3279 cm−1) and carbonyl (1610 cm−1)
1
groups. Its H NMR data (Table 2) exhibited signals for 1,2-
disubstituted benzene protons at δH 8.22 (1H, d, J = 7.9 Hz),
7.64 (1H, t, J = 8.5 Hz), 7.46 (1H, d, J = 8.4 Hz), and 7.37
(1H, t, J = 7.4 Hz), 1,3,4-trisubstituted benzene protons at δH
6.82 (1H, d, J = 8.5 Hz), 6.75 (1H, br d, J = 8.5 Hz), and 6.73
(1H, br s), two mutually coupled methylene protons at δH 3.14
F
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Figure 3. Comparative analysis of the experimental and computational ECD spectra (shift = 16 nm; σ = 0.30) of compounds 1−4 and 6 (A and B)
and the assignment of the absolute configuration of 5 (2R,3R,7″S,8″S) by comparing the spectra to that of compound 4 (2S,3S,7″S,8″S) (C).
Comparison of the experimental and computational ECD spectra (shift: 0 nm; σ = 0.1) of compounds 7−9 (D).
compounds, rhamnoneuronal C (6) and balansechromanones
A (7) and B (8) showed the greatest potential in regard to
senolytic activity. In particular, the cell viability of the
rhamnoneuronal C (6)-treated group was reduced by 34% in
the senescent cells compared with the cell viability of the
control, thereby showing the most specific senolytic property
(Figure 4B). Additionally, a luciferase reporter assay system
was utilized with the newly isolated compounds to evaluate
their ability to inhibit the promoter activity of p16INK4A,
which is a specific marker in senescent cells. After treatment
with the compounds, rhamnoneuronal C (6) significantly
reduced the promoter activity of p16INK4A by 32%. Thus,
rhamnoneuronal C (6) may be expected to possess
p16INK4A-dependent senolytic activity (Figure 4C).
In conclusion, the present study proposes new senolytic
candidates that selectively remove senescent cells to reduce
their harmful effects on normal replicating HDF cells. In the
cell viability comparison experiment between young and aged
cells to determine the senolytic properties of the isolates,
compounds 6−8 were shown to have potential for use as lead
compounds of senolytic agents. Furthermore, the transcrip-
tional activation of the p16INK4A promoter, a representative
senescence marker, was reduced markedly only with
compound 6, whereas compounds 7 and 8 do not show this
reduction. From these observations, it is likely that compounds
6 and 7 (or compound 8) exert senolytic properties via
different modes of action. Since a direct link based on
convincing evidence between cell death and the inhibition of
p16INK4A has not been fully established until now, further
studies on the relationship between p16INK4A and cell death
will be needed. Taken together, compounds 6−8 may have use
as new senolytic agent leads and hence contribute to relevant
mechanistic studies.
EXPERIMENTAL SECTION
■
General Experimental Procedures. Optical rotations were
acquired with a JASCO P-2000 polarimeter (JASCO, Tokyo,
Japan). UV and ECD spectra were obtained using a Chirascan-Plus
CD spectrometer (Applied Photophysics, Leatherhead, UK). IR
spectra were recorded using a JASCO FT/IR-4200 spectrometer.
NMR data were measured using a JNM-ECZ-400S (JEOL, Tokyo,
Japan) and Bruker Avance-800 MHz spectrometer (Bruker, Billerica,
MA, USA). HRESIMS were acquired with an Agilent 6530 Q-TOF
mass spectrometer (Agilent Technologies, Santa Clara, CA, USA) and
a Waters Xevo G2 QTOF mass spectrometer (Waters, Milford, MA,
USA). Zeoprep silica gel (63−200 μm, Zeochem, uetikon am See,
Switzerland), Cosmosil 75C18-OPN (75 μm, Nacalai Tesque, Kyoto,
Japan), and Sephadex LH-20 (Sigma-Aldrich, St. Louis, MO, USA)
were used for column chromatography. Thin-layer chromatography
(TLC) analyses were performed with TLC silica gel 60 F254 and TLC
silica gel 60 RP-18 F254S plates (Merck, Darmstadt, Germany).
Preparative HPLC was carried out using a Gilson system (Gilson,
Villiers-le-Bel, France) with a UV detector at 205 or 254 nm and an
Optima Pak C18 column (10 × 250 mm, 10 μm, RS Tech, Seoul,
Korea).
Plant Material. The plant material was collected from the Ha Vi
commune, Bach Thong district, Bac Kan province, Vietnam
(22°13′08.3″ N, 105°50′05.4″ E), in April 2018. Based on
morphological characteristics, the samples were identified as
G
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Figure 4. Compounds 6−8 selectively removed targeted senescent cells. Proliferating (A) or senescent human dermal fibroblast cells (B) were
exposed to 20 μM of the various isolated compounds for 3 days. The cell viability percentages were evaluated using the MTT method. The effect of
12 different compounds on the promoter activity of p16INK4A in human dermal fibroblasts (C). Compounds 1−12 were exposed to cells for 24 h,
and the activity of the p16INK4A promoter was shown by comparing the compound-treated groups to the untreated group.
Rhamnoneuron balansae (Drake) Gilg by Professor Duc Trong
Nghiem, Department of Botany, Hanoi University of Pharmacy,
Hanoi, Vietnam. A voucher specimen was deposited in the Medicinal
Herbarium of Hanoi University of Pharmacy (HNIP) with the
accession number HNIP.18510/15.
(R4.10.1−R4.10.6). Fraction R4.10.6 (70 mg) was purified by
HPLC and eluted with MeOH/H2O (3:7, 2 mL/min) to obtain
compounds 2 (2.1 mg) and 6 (2.0 mg). Fraction R4.12 was separated
by Sephadex LH-20 CC and eluted with MeOH to afford five
subfractions (R4.12.1−R4.12.5). Fraction R4.12.3 (35 mg) was
purified by HPLC and eluted with MeOH/H2O (4:6, 2 mL/min)
to obtain compound 1 (5.0 mg).
Extraction and Isolation. The roots of R. balansae (30 kg) were
extracted with 70% EtOH (3 × 10 L), and the resulting extract was
freeze-dried to yield a brown powder (730 g). This extract was
suspended in water and sequentially partitioned with n-BuOH (3 × 4
L). The n-BuOH-soluble part (220 g) was preadsorbed on silica gel
for column chromatography and eluted with an n-hexane and EtOAc
gradient (2:1 → 0:1, v/v) to yield four fractions (R1−R4). Fraction
R2 (10 g) was separated into 10 fractions (R2.1−R2.10) via RP-C18
column chromatography and eluted with a MeOH/H2O gradient (3:7
→ 1:0, v/v). Fraction R2.6 (1.0 g) was subjected to Sephadex LH-20
column chromatography (CC) and eluted with MeOH to afford six
subfractions (R2.6.1−R2.6.6). Fraction R2.6.3 was purified by
preparative HPLC with CH3CN/H2O (3:7, 2 mL/min) to afford
compounds 10 (15.1 mg), 11 (8.2 mg), and 12 (10.5 mg). Fraction
R2.8 (1.2 g) was separated by Sephadex LH-20 CC and eluted with
MeOH to obtain seven subfractions (R2.8.1−R2.8.7). Fraction R2.8.2
(200 mg) was purified by HPLC and eluted with CH3CN/H2O (4:6,
2 mL/min) to obtain compounds 13 (10 mg) and 14 (5.0 mg).
Fraction R2.8.4 was purified by preparative HPLC with MeOH/H2O
(7:3, 2 mL/min) to afford compounds 7 (1.7 mg), 8, (3.0 mg), and 9
(3.0 mg). Fraction R2.8.5 (70 mg) was purified by HPLC and eluted
with MeOH/H2O (6:4, 2 mL/min) to obtain compound 15 (5.7
mg). Fraction R4 (120 g) was separated into 19 fractions (R4.1−
R4.19) via RP-C18 CC and eluted with a MeOH/H2O gradient (2:8
→ 1:0, v/v). Fraction R4.8 was separated by Sephadex LH-20 CC and
eluted with MeOH to afford four subfractions (R4.8.1−R4.8.4).
Fraction R4.8.3 (50 mg) was purified by HPLC and eluted with
MeOH/H2O (3:7, 2 mL/min) to obtain compounds 3 (3.0 mg), 4
(3.1 mg), and 5 (2.0 mg). Fraction R4.10 was separated by Sephadex
LH-20 CC and eluted with MeOH to afford six subfractions
Rhamnoneuronal A (1): yellowish powder; [α]2D0 −15 (c 1.0,
MeOH); UV (MeOH) λmax (log ε) 195 (3.96), 225 (3.82), 285
(3.21) nm; IR νmax 3375, 2947, 2835, 1659, 1618, 1450, 1415, 1114,
1026, 693 cm−1; 1H and 13C NMR data, see Tables 1 and 3;
HRESIMS m/z 513.1192 [M − H]− (calcd for C29H21O9, 513.1191).
Rhamnoneuronal B (2): yellowish powder; [α]2D0 −52 (c 1.0,
MeOH); UV (MeOH) λmax (log ε) 200 (3.91), 225 (3.71), 285
(3.12) nm; IR νmax 3357, 1612, 1517, 1454, 1255, 1073, 1019 cm−1;
1H and 13C NMR data, see Tables 1 and 3; HRESIMS m/z 529.1117
[M − H]− (calcd for C29H21O10, 529.1140).
Rhamnoneuroside A (3): yellowish powder; [α]2D0 −98 (c 1.0,
MeOH); UV (MeOH) λmax (log ε) 195 (3.96), 225 (3.67), 285
(3.08) nm; IR νmax 3389, 1600, 1358, 1055, 1012, 835, 671 cm−1; 1H
and 13C NMR data, see Tables 1 and 3; HRESIMS m/z 675.1716 [M
− H]− (calcd for C35H31O14, 675.1714).
Rhamnoneuroside B (4): yellowish powder; [α]2D0 −140 (c 1.0,
MeOH); UV (MeOH) λmax (log ε) 195 (3.96), 225 (3.67), 285
1
(3.08) nm; IR νmax 3347, 1616, 1085, 1028, 835, 687 cm−1; H and
13C NMR data, see Tables 1 and 3; HRESIMS m/z 675.1724 [M −
H]− (calcd for C35H31O14, 675.1719).
Rhamnoneuroside C (5): yellowish powder; [α]2D0 −45 (c 1.0,
MeOH); UV (MeOH) λmax (log ε) 195 (3.95), 230 (3.65), 285
(3.09) nm; IR νmax 3361, 1617, 1079, 982, 836, 685 cm−1; 1H and 13
C
NMR data, see Tables 1 and 3; HRESIMS m/z 675.1716 [M − H]−
(calcd for C35H31O14, 675.1714).
Rhamnoneuronal C (6): yellowish powder; [α]2D0 −10 (c 1.0,
MeOH); UV (MeOH) λmax (log ε) 195 (3.94), 225 (3.84), 300
1
(3.48) nm; IR νmax 3336, 1611, 1517, 1054, 1032, 671 cm−1; H and
H
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Article
13C NMR data, see Tables 1 and 3; HRESIMS m/z 511.1028 [M −
H]− (calcd for C29H19O9, 511.1035).
HyClone) in an atmosphere of 5% CO2 and incubated at 37 °C.
Cellular senescence was induced by the replication of passages. After
the cells were cultured to passage 35, the cellular senescence level was
confirmed by monitoring the colorimetric analysis of the senescence-
associated β-galactosidase and mRNA expression level of p16INK4A
(Figure S103, Supporting Information).
Cell Viability Assay. The cytotoxicity of compounds was
evaluated using an MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenylte-
trazolium bromide) assay (Sigma, MO, USA). HDF cells were seeded
at a density of 5 × 103 cells/well in 96-well plates and incubated for
24 h to attach cells before being treated with the isolated compounds.
Then, the cells were treated with the test compounds at a
concentration of 20 μM and incubated for 3 days. To avoid the
solvent toxicity of dimethyl sulfoxide (DMSO) in the medium, the
final DMSO concentration was kept at 0.05%. Then, 20 μL of a 2 mg/
mL MTT solution was added to each well, followed by incubation for
2 h. To measure the absorbance, a Versamax microplate reader
(VersaMax, PA, USA) was used at 570 nm.16
Luciferase Reporter Assay. Reporter gene transfection was
performed using Lipofectamine LTX (Thermo Fisher Scientific) in
HDF cells. Prior to the experiment, transfection efficiency was
measured using a GFP-tagged plasmid. Subsequently, HDF cells were
cultured at a density of 3 × 104 cells/well in 24-well plates. After 24 h,
the medium was transferred to fresh DMEM supplemented only with
10% FBS. The pGL3_p16INK4A promoter (0.4 μg), which encodes
firefly luciferase driven by the p16INK4A promoter and β-
galactosidase (0.1 μg, encoding Renilla luciferase in 25 μL of Opti-
MEM), was incubated with Lipofectamine LTX (0.75 μL, Invitrogen,
Carlsbad, CA, USA) and PLUS reagent (0.25 μL, Invitrogen) in 25
μL of Opti-MEM (Gibco/Thermo Fisher Scientific, Waltham, MA,
USA) for 10 min at room temperature. Then, 50 μL of the DNA−
lipid complex was added to each well. The mixture was exposed to the
cells for 5 h and incubated overnight with DMEM supplemented with
10% FBS. Next, the cells were treated with DMSO or the isolated
components in a serum-free medium for an additional 24 h. The
luciferase activity was determined as the percentage of firefly/Renilla
luciferase activity. The activity of the p16INK4A promoter in the
presence of compounds relative to that of the vehicle is shown.16
Statistical Analysis. Data are expressed as the means standard
deviations (SDs) of three independent experiments and were
calculated and plotted by one-way analysis of variance (ANOVA).
Statistically significant differences are indicated as the following: *p <
0.05, **p < 0.01, and ***p < 0.001.
Balansechromanone A (7): yellowish powder; [α]2D0 +83 (c 1.0,
MeOH); UV (MeOH) λmax (log ε) 195 (4.51), 215 (4.29), 250
1
(3.71) nm; IR νmax 3361, 1607, 1465, 1226, 831, 764, 662 cm−1; H
and 13C NMR data, see Tables 2 and 3; HRESIMS m/z 267.1015 [M
− H]− (calcd for C17H15O3, 267.1021).
Balansechromanone B (8): yellowish powder; [α]2D0 +101 (c 1.0,
MeOH); UV (MeOH) λmax (log ε) 195 (4.51), 215 (4.29), 250
(3.71) nm; IR νmax 3426, 1687, 1606, 1514, 1463, 1228, 1150, 1032,
1
882, 765, 632 cm−1; H and 13C NMR data, see Tables 2 and 3;
HRESIMS m/z 299.1281 [M − H]+ (calcd for C18H19O4, 299.1283).
Balansechromanone C (9): yellowish powder; [α]2D0 +85 (c 1.0,
MeOH); UV (MeOH) λmax (log ε) 200 (4.62), 220 (4.63), 250
(3.23) nm; IR νmax 3362, 1671, 1605, 1464, 1314, 1150, 882, 615
1
cm−1; H and 13C NMR data, see Tables 2 and 3; HRESIMS m/z
267.1012 [M − H]− (calcd for C17H15O3, 267.1021).
Balansechromone A (10): yellowish powder; [α]2D0 +60 (c 1.0,
MeOH); UV (MeOH) λmax (log ε) 200 (4.56), 235 (4.54), 283
(4.04), 320 (3.80) nm; IR νmax 3279, 1610, 1515, 1215, 811, 616
1
cm−1; H and 13C NMR data, see Tables 2 and 3; HRESIMS m/z
311.0913 [M − H]− (calcd for C18H15O5, 311.0919).
Balansechromone B (11): yellowish powder; [α]2D0 +64 (c 1.0,
MeOH); UV (MeOH) λmax (log ε) 200 (4.43), 235 (4.47), 283
(4.03), 320 (3.90) nm; IR νmax 3362, 1618, 1515, 1211, 1032, 630
1
cm−1; H and 13C NMR data, see Tables 2 and 3; HRESIMS m/z
327.1232 [M + H]+ (calcd for C18H19O5, 327.1232).
Balansechromone C (12): yellowish powder; [α]2D0 +63 (c 1.0,
MeOH); UV (MeOH) λmax (log ε) 200 (4.54), 235 (4.40), 283
(4.01), 320 (3.82) nm; IR νmax 3331, 1617, 1467, 1232, 759, 700
1
cm−1; H and 13C NMR data, see Tables 2 and 3; HRESIMS m/z
295.0977 [M − H]− (calcd for C18H15O4, 295.0970).
Sugar Analysis. The absolute configurations of the mono-
saccharide units present in the isolated compounds were analyzed
with the same procedures as in a previous investigation.33 Detailed
information is shown in the Supporting Information (Figure S100).
Preparation of the (S)-MTPA and (R)-MTPA Ester Deriva-
tives of Compound 1. Compound 1 (1.0 mg, 1.95 μmol) was
dissolved in dry pyridine (489.3 μL, 6.05 μmol, 3.1 equiv), and then
anhydrous CHCl3 was added to the residue. S-(+)-MTPA-Cl (0.73
μL, 3.9 μmol, 2.0 equiv) and 4-dimethylaminopyridine (0.3 mg) were
added to the mixture and stirred at room temperature for 3 h. The
reaction solution was checked by TLC analysis and fractionated three
times with ethyl acetate and water. The ethyl acetate extract was dried
1
using an evaporator, and the H NMR data were directly recorded
ASSOCIATED CONTENT
■
due to the small scale of the reaction. The above step was repeated
sı
* Supporting Information
1
using R-(−)-MTPA-Cl to give the H NMR spectrum of the (S)-
The Supporting Information is available free of charge at
https://pubs.acs.org/doi/10.1021/acs.jnatprod.0c00885.
MTPA ester derivative. The use of the Mosher method was based on
a previous paper.24
Computational ECD Analysis. The computational analysis of
compounds 1 and 7 was performed using MMFF94 calculations with
a search limit of 1.0 kcal/mol in Conflex 7 (ConflexCorp., Tokyo,
Japan). We used TmoleX 4.4 and Turbomole to optimize the ground-
state geometry of the main conformers using the def-SV (P) basis set
and the B3LYP functional levels with compounds 1 and 7. The
computed ECD spectra of all optimized conformers were represented
by TDDFT using the 6-31G basis set and the B3LYP functional levels
for compound 1 and the def-SV(P) basis set and the B3LYP
functional levels for compound 7 through Boltzmann distributions.
The detailed calculation method was performed using the same
process as reported in a previous paper.33 All calculated ECD spectra
were generated with a UV shift of 16 nm and σ = 0.3 eV for
compound 1 and without a UV shift and σ = 0.1 for 7 using Specdis
NMR, HRESIMS, and IR spectra of compounds 1−12;
1H NMR spectra of the R- and S-MTPA esters of 1; 3D
structures of the R- and S-MTPA esters; and detailed in
silico predicted ECD data of compounds 1 and 7 (PDF)
AUTHOR INFORMATION
■
Corresponding Author
Won-Keun Oh − Korea Bioactive Natural Material Bank,
Research Institute of Pharmaceutical Sciences, College of
Pharmacy, Seoul National University, Seoul 08826, Republic
of Korea; orcid.org/0000-0003-0761-3064; Phone: +82-
2-880-7872; Email: wkoh1@snu.ac.kr
1.70.1 (University of Wurzburg, Wurzburg, Germany).
̈ ̈
Cell Culture. A primary culture of human dermal fibroblast cells
was kindly provided by Professor Kyung A. Cho of Chonnam
National University. HDF cells were maintained under subconfluent
conditions in Dulbecco’s modified Eagle’s medium (DMEM,
HyClone, Logan, UT, USA) supplemented with 10% fetal bovine
serum (FBS, HyClone) and 1% penicillin/streptomycin (P/S,
Authors
Hyo-Moon Cho − Korea Bioactive Natural Material Bank,
Research Institute of Pharmaceutical Sciences, College of
Pharmacy, Seoul National University, Seoul 08826, Republic
of Korea; orcid.org/0000-0003-3447-8726
I
https://dx.doi.org/10.1021/acs.jnatprod.0c00885
J. Nat. Prod. XXXX, XXX, XXX−XXX

Journal of Natural Products
pubs.acs.org/jnp
Article
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Yae-Rin Lee − Korea Bioactive Natural Material Bank,
Research Institute of Pharmaceutical Sciences, College of
Pharmacy, Seoul National University, Seoul 08826, Republic
of Korea
Ba-Wool Lee − Korea Bioactive Natural Material Bank,
Research Institute of Pharmaceutical Sciences, College of
Pharmacy, Seoul National University, Seoul 08826, Republic
of Korea; orcid.org/0000-0002-4944-5145
Mi Zhang − Korea Bioactive Natural Material Bank, Research
Institute of Pharmaceutical Sciences, College of Pharmacy,
Seoul National University, Seoul 08826, Republic of Korea
Byeol Ryu − Korea Bioactive Natural Material Bank,
Research Institute of Pharmaceutical Sciences, College of
Pharmacy, Seoul National University, Seoul 08826, Republic
of Korea; orcid.org/0000-0002-3405-2875
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Du-Trong Nghiem − Department of Botany, Hanoi
University of Pharmacy, Hanoi, Vietnam
Ha-Thanh-Tung Pham − Department of Botany, Hanoi
University of Pharmacy, Hanoi, Vietnam
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Complete contact information is available at:
https://pubs.acs.org/10.1021/acs.jnatprod.0c00885
(18) Xu, M.; Palmer, A. K.; Ding, H.; Weivoda, M. M.; Pirtskhalava,
T.; White, T. A.; Sepe, A.; Johnson, K. O.; Stout, M. B.; Giorgadze,
N.; Jensen, M. D.; LeBrasseur, N. K.; Tchkonia, T.; Kirkland, J. L.
eLife 2015, 4, e12997.
Notes
The authors declare no competing financial interest.
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McGowan, S. J.; Fuhrmann-Stroissnigg, H.; Gurkar, A. U.; Zhao, J.;
Colangelo, D.; Dorronsoro, A.; Ling, Y. Y.; Barghouthy, A. S.;
Navarro, D. C.; Sano, T.; Robbins, P. D.; Niedernhofer, L. J.;
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ACKNOWLEDGMENTS
■
This research was funded by grants from the Korea Bioactive
Natural Material Bank (NRF-2017M3A9B8069409) and from
the Basic Science Research Program (NRF-2017R1E1A1-
A01074674) through the National Research Foundation of
Korea funded by the Ministry of Science, ICT and Planning.
We thank Dr. Hoang Minh Chau, CEO of the Nam Duoc
Pharmaceutical Joint Stock Company in Vietnam, for 70%
EtOH extraction and freeze-drying of R. balansae roots.
(23) Liu, F.; Li, F. S.; Feng, Z. M.; Yang, Y. N.; Jiang, J. S.; Li, L.;
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https://dx.doi.org/10.1021/acs.jnatprod.0c00885
J. Nat. Prod. XXXX, XXX, XXX−XXX