Benzofuran and coumarin derivatives from the root of Angelica dahurica and their PPAR-γ ligand-binding activity

Article abstract of DOI:10.1016/j.phytochem.2020.112301

A phytochemical investigation of the root of Angelica dahurica led to the isolation of benzofuran and coumarin derivatives. This is the first report of the isolation and identification of three furanocoumarin sulfates from A. dahurica root. The structures of a total of twelve undescribed compounds were determined by extensive spectroscopic analysis, including 2D NMR data, hydrolysis, and solvolysis, followed by either physicochemical and spectroscopic data or X-ray crystallographic analysis. The isolated compounds were evaluated for their PPAR-γ ligand-binding activity, and six compounds showed significant PPAR-γ ligand-binding activity. In particular, the undescribed benzofuran derivative, 3-[6,7-furano-9-hydroxy-4-(2″,3″-dihydroxy-3″-methylbutyloxy)]-phenyl propionic acid, exhibited the most potent PPAR-γ ligand-binding activity and accumulated intracellular lipid in 3T3-L1 cells.

Full text of DOI:10.1016/j.phytochem.2020.112301

Phytochemistry173(2020)112301
Contents lists available at ScienceDirect
Phytochemistry
journal homepage: www.elsevier.com/locate/phytochem
Benzofuran and coumarin derivatives from the root of Angelica dahurica and
their PPAR-γ ligand-binding activity
Yukiko Matsuo∗, Emi Yamaguchi, Ryo Hakamata, Kanae Ootomo, Kazuhiro Takatori,
Haruhiko Fukaya, Yoshihiro Mimaki
Tokyo University of Pharmacy and Life Sciences, School of Pharmacy, 1432-1 Horinouchi, Hachioji, Tokyo, 192-0392, Japan
A R T I C L E I N F O
A B S T R A C T
Keywords:
A phytochemical investigation of the root of Angelica dahurica led to the isolation of benzofuran and coumarin
derivatives. This is the first report of the isolation and identification of three furanocoumarin sulfates from A.
dahurica root. The structures of a total of twelve undescribed compounds were determined by extensive spec-
troscopic analysis, including 2D NMR data, hydrolysis, and solvolysis, followed by either physicochemical and
spectroscopic data or X-ray crystallographic analysis. The isolated compounds were evaluated for their PPAR-γ
ligand-binding activity, and six compounds showed significant PPAR-γ ligand-binding activity. In particular, the
undescribed benzofuran derivative, 3-[6,7-furano-9-hydroxy-4-(2″,3″-dihydroxy-3″-methylbutyloxy)]-phenyl
propionic acid, exhibited the most potent PPAR-γ ligand-binding activity and accumulated intracellular lipid in
3T3-L1 cells.
Angelica dahurica
Apiaceae
X-ray crystallographic analysis
Benzofuran
Coumarin
Furanocoumarin
PPAR-γ
3T3-L1 cells
1. Introduction
2. Results and discussion
Angelica dahurica (Hoffm.) Benth. & Hook.f. ex Franch. & Sav.
(Apiaceae) is distributed throughout Northeast China and Japan, and its
root has long been used as a traditional medicine for the treatment of
erythema, headache, and inflammation (Saruwatari et al., 2014). The
root of A. dahurica is known to contain furanocoumarins, benzofurans,
and polyacetylenes that are conserved, specialized metabolites in plants
belonging to the Apiaceae family (Oh et al., 2002). The constituents
isolated from A. dahurica root have been reported to exhibit various
biological activities such as β-secretase inhibitory activity, anti-
microbial activity, and peroxisome proliferator-activated receptor
(PPAR)-γ ligand-binding activity (Jiang et al., 2019; Kwon et al., 1997;
Liu et al., 2011; Marumoto and Miyazawa, 2010). As part of our search
for bioactive specialized products from higher plants, a chemical ex-
amination of the methanol (MeOH) extract of A. dahurica root was
conducted, which resulted in the isolation of twelve undescribed and
nine known benzofuran and coumarin derivatives. Here, we report the
structural characteristics of the undescribed compounds, which were
determined by extensive spectroscopic analysis, including two-dimen-
sional (2D) NMR, hydrolysis, and solvolysis, followed by either physi-
cochemical and spectroscopic data or X-ray crystallographic analysis.
PPAR-γ ligand-binding activity of the isolated compounds is also dis-
cussed.
The MeOH extract of A. dahurica root was passed through a porous
polymer resin (Diaion® HP-20) column. The MeOH- and 50% MeOH-
eluted fractions were repeatedly subjected to column chromatography
(CC) on silica gel and octadecylsilanized (ODS) columns, as well as
preparative ODS HPLC to obtain 21 compounds (1–21) (Fig. 1). Com-
pounds 4–6, 8, 10–11, 16–17, and 20 were identified as cnidioside A
(Chen et al., 2001) (4), methylcnidioside A (Chen et al., 2001) (5),
methylpicraquassioside (Chen et al., 2001) (6), 1′-O-β-D-glucopyr-
anosyl-3′-hydroxynodakenetin (Niu et al., 2002) (8), columbianetin 2′-
O-β-D-glucopyranoside (Kim et al., 2010) (10), columbianetin 2′-O-β-D-
apiofuranosyl-(1 → 6)-β-D-glucopyranoside (VanWagenen et al., 1988)
(11), byakangelicin 3″-O-β-D-glucopyranoside (Thastrup and Lemmich,
1983) (16), byakangelicin 3″-O-β-D-apiofuranosyl-(1 → 6)-β-D-gluco-
pyranoside (Jia et al., 2008) (17), and 5,8-bis-(2,3-dihydroxy-3-me-
thylbutyloxy)-psoralen (Kwon et al., 1997) (20).
Compound 1 was isolated as an amorphous solid ([α]D +26.0 in
MeOH) with the molecular formula C16H20O7, as determined by high-
resolution electrospray ionization time-of-flight mass spectrometry
(HR-ESI-TOF-MS; m/z 347.1107 [M + Na]+, calcd. 347.1107) and 13C
NMR (16 carbon signals) spectral data. The UV spectrum of 1 had ab-
sorption maxima at 283 (log ε 3.55) and 255 (log ε 3.89) nm, and the IR
spectrum showed absorption bands attributed to hydroxy groups at
∗ Corresponding author.
E-mail address: matsuoy@toyaku.ac.jp (Y. Matsuo).
https://doi.org/10.1016/j.phytochem.2020.112301
Received 8 November 2019; Received in revised form 10 February 2020; Accepted 12 February 2020
0031-9422/©2020ElsevierLtd.Allrightsreserved.

Y. Matsuo, et al.
Phytochemistry173(2020)112301
Fig. 1. Structures of 1–21 isolated from the root of Angelica dahurica.
3362 cm−1, a carbonyl group at 1714 cm−1, and an aromatic ring at
1558 and 1455 cm−1. The 1H and 13C NMR spectra of 1 were similar to
those of the aglycone of 4, showing signals for a pair of olefinic protons
at δH 7.46 (d, J = 2.2 Hz, H-2′) and 6.90 (dd, J = 2.2, 0.8 Hz, H-3′); an
aromatic proton at δH 6.64 (d, J = 0.8 Hz, H-8); an ethylene group at δH
2.98 (2H, dt, J = 14.0, 6.9 Hz, H2-3) and δH 2.51 (2H, t, J = 6.9, H2-2);
an aromatic carbon with a hydroxy group at δC 155.5 (C-9); and the
carbonyl carbon of a carboxy group at δC 182.9 (C-1). In addition, the
1H and 13C NMR spectra were indicative of the presence of a C5 unit
comprising an oxymethylene [δH 4.47 (dd, J = 9.8, 2.5 Hz, H-1″a) and
4.16 (dd, J = 9.8, 8.3 Hz H-1″b)/δC 75.2 (C-1″)]; a hydroxymethine [δH
3.80 (dd, J = 8.3, 2.5 Hz, H-2″)/δC 78.5 (C-2″)]; two methyl groups [δH
1.28 and 1.22 (each 3H, s, Me-4″ and Me-5″)/δC 26.8 and 25.0 (C-4″
and C-5″)]; and an oxygenated quaternary carbon [δC 72.7 (C-3″)] in
the molecule of 1. The long-range correlations from Me-4″ and Me-5″ to
C-2″ and C-3″, and from H2-1″ to C-5 in the 1H-detected heteronuclear
multiple-bond connectivity (HMBC) spectrum of 1, as shown in Fig. 2,
allowed the C5 unit to be identified as a 2,3-dihydroxy-3-methylbuty-
loxy group linked to C-5. Compound 1 was thus established as 3-[6,7-
furano-9-hydroxy-4-(2″,3″-dihydroxy-3″-methylbutyloxy)]-phenyl pro-
pionic acid. The absolute configuration at C-2″ remains to be de-
termined.
Based on the HR-ESI-TOF-MS and 13C NMR data, 2 was shown to
have the molecular formula of C22H30O12, which was larger than that of
1 by C6H10O5, corresponding to one hexosyl unit. The 1H and 13C NMR
spectra of 2 indicated the presence of a β-D-glucopyranosyl unit (Glc)
[δH 4.89 (d, J = 8.5 Hz); δC 103.4 (CH), 74.8 (CH), 77.9 (CH), 71.4
(CH), 78.1 (CH), 62.6 (CH2)]. Enzymatic hydrolysis of 2 with β-D-
glucosidase gave 1 and D-glucose. In the HMBC spectrum of 2, a long-
range correlation was observed between H-1 of Glc and C-9 at δC 155.8.
2

Y. Matsuo, et al.
Phytochemistry173(2020)112301
the signals attributable to a β-D-glucopyranosyl unit glycosylated at C-6
[δH 5.28 (d, J = 7.5 Hz); δC 98.7 (CH), 74.8 (CH), 78.5 (CH), 71.5 (CH),
76.6 (CH), 68.1 (CH2)] and a terminal β-D-apiofuranosyl unit (Api) [δH
5.64 (d, J = 2.5 Hz); δC 110.9 (CH), 77.8 (CH), 80.4 (C), 75.0 (CH2),
65.6 (CH2)] were observed. In the HMBC spectrum of 9, long-range
correlations were observed between H-1 of Api and C-6 of Glc at δC 68.1
and between H-1 of Glc and C-4′ of the aglycone at δC 78.6. Compound
9
was established as (3′S)-hydroxy-nodakenetin 4′-O-β-D-apiofur-
anosyl-(1 → 6)-β-D-glucopyranoside.
Compound 12 had the molecular formula C28H36O16, and its 1H
NMR spectrum contained signals assignable to a furanocoumarin
moiety at δH 8.47 and 6.28 (each d, J = 9.8 Hz, H-4 and H-3); 7.79 and
7.26 (each d, J = 2.4 Hz, H-2′ and H-3′); and 7.17 (s, H-8).
Furthermore, the 1H and 13C NMR signals attributable to a 2,3-dihy-
droxy-3-methylbutyloxy moiety [δH 4.95 (dd, J = 9.7, 2.3 Hz, H-1″a)
and 4.40 (dd, J = 9.7, 9.0 Hz, H-1″b)/δC 75.3 (C-1″); δH 4.13 (dd,
J = 9.0, 2.3 Hz, H-2″)/δC 75.8 (C-2″); δC 77.9 (C-3″), and δH 1.35 and
1.25 (each 3H, s, Me-4″ and Me-5″)/δC 23.2 and 21.7 (C-4″ and C-5″)];
and a β-D-fructofuranosyl-(2 → 1)-α-D-glucopyranoside (sucrose, Fru-
(2 → 1)-Glc) unit [δH 3.50 and 3.46 (each d, J = 12.3 Hz, Fru-H2-1)/δC
64.0 (Fru-C-1), 105.1 (Fru-C-2), 79.2 (Fru-C-3), 76.7 (Fru-C-4), 82.5
(Fru-C-5), 65.2 (Fru-C-6); δH 5.19 (d, J = 3.9 Hz, Glc-H-1)/δC 93.1 (Glc-
C-1), 73.2 (Glc-C-2), 74.8 (Glc-C-3), 71.7 (Glc-C-4), 74.4 (Glc-C-5), 62.8
(Glc-C-6)] were observed. Acid hydrolysis of 12 gave R-(+)-oxypeu-
cedanin hydrate (Fujioka et al., 1999) (12a), D-glucose, and D-fructose.
The Fru-C-6 signal of the sucrose unit was shifted downfield by ap-
proximately 2 ppm compared to that of authentic sucrose, and Fru-H2-
6 at δH 3.90 (dd, J = 9.9, 7.9 Hz) and 3.68 (dd, J = 7.9, 2.4 Hz) ex-
hibited HMBC correlations with C-3″ of the furanocoumarin moiety at
δC 77.9. Based on the above data, 12 was defined as R-(+)-oxypeuce-
danin hydrate sucrose ether, as shown in Fig. 1.
Fig. 2. Key HMBC correlations of 1.
Compound 2 was assigned as 3-[6,7-furano-9-(β-D-glucopyranosyloxy)-
4-(2″,3″-dihydroxy-3″-methylbutyloxy)]-phenyl propionic acid.
Compound 3 (C23H32O12) was presumed to be a methyl ester of 2
based on the 1H and 13C NMR data. The deshielded methyl proton
signal at δH 3.65 (s) had an HMBC correlation with the C-1 carbonyl
carbon at δC 176.6. Thus, 3 was identified as 3-[6,7-furano-9-(β-D-
glucopyranosyloxy)-4-(2″,3″-dihydroxy-3″-methylbutyloxy)]-phenyl
propionic acid methyl ester.
Compound 7 was isolated as a triclinic crystal with a molecular
formula of C14H16O6. The 1H and 13C NMR spectra of 7 were typical
spectra of a 6,7-disubstituted coumarin derivative, showing signals for
(Z)-alkene protons at δH 7.83 (d, J = 9.4 Hz, H-4) and 6.16 (d,
J = 9.4 Hz, H-3) as well as for two aromatic protons at δH 7.40 (s, H-5)
and 6.72 (s, H-8). The downfield-shifted aromatic carbon at δC 161.5
was assigned to C-7 and was indicative of the presence of a hydroxy
group at C-7. Furthermore, a C5 unit comprising of a hydroxymethine
[δH 3.82 (dd, J = 10.5, 2.3 Hz, H-2′)/δC 76.1 (C-2′)]; a hydro-
xymethylene [δH 3.62 and 3.52 (each d, J = 10.8 Hz, H2-4′)/δC 68.6 (C-
4′)]; a methylene [δH 3.12 (dd, J = 14.1, 2.3 Hz, H-1′a) and 2.64 (dd,
J = 14.1, 10.5 Hz, H-1′b)/δC 32.8 (C-1′)]; a methyl [δH 1.20 (s, Me-5′)/
δC 19.1 (C-5′)]; and an oxygenated quaternary carbon [δC 75.5 (C-3′)]
was shown to be present in the molecule of 7. The HMBC correlations,
as shown in Fig. 3, allowed the C5 unit to be identified as a 2,3,4-tri-
hydroxy-3-methylbutyloxy group linked to C-6. Accordingly, 7 was
defined as 7-hydroxy-6-(2′,3′,4′-trihydroxy-isopentanyl)-coumarin.
Compound 7 did not show specific rotation, and single-crystal X-ray
crystallographic analysis of 7 gave racemic crystals in the space group
P-1. Thus, 7 was revealed to be a racemate.
Compound 13 had the same molecular formula as 12 (C28H36O16),
and its 1H and 13C NMR spectra were quite similar to those of 12,
suggesting that R-(+)-oxypeucedanin hydrate contained a sucrose unit
(Fujioka et al., 1999). In the HMBC spectrum of 13, Glc-H-3 at δH 3.97
(dd, J = 9.5, 8.9 Hz) of the sucrose unit exhibited a long-range cor-
relation with C-3″ of the furanocoumarin moiety at δC 79.2. Acid hy-
drolysis of 13 gave 12a, D-glucose, and D-fructose. Thus, 13 was de-
termined to be an isomer of 12 with regard to the sucrose linkage
position to R-(+)-oxypeucedanin hydrate, as shown in Fig. 1.
Compound 14 had the molecular formula of C16H16O9S, as revealed
by HR-ESI-TOF-MS (m/z: 407.0408 [M + Na]+), and its 1H and 13C
NMR spectra suggested the structure of an oxypeucedanin hydrate de-
rivative. The IR spectrum of 14 showed absorption bands for a hydroxy
group at 3360 cm−1 and for a sulfate group at 1349 and 1097 cm−1
.
Solvolysis of 14 in pyridine-dioxane at 100 °C gave ( )-oxypeuce-
danin hydrate (Fujioka et al., 1999) (14a) and an aqueous solution
containing sulfuric acid, which was evident from the white precipitate
(BaSO4) obtained on the addition of an aqueous solution of BaCl2 (Kim
et al., 2010; Yang et al., 2016). These results implied that 14 was a
sulfate of 14a. When the 1H and 13C NMR spectra of the two were
Compound 9 had the molecular formula C25H32O14, and its 1H and
13C NMR spectral features were closely related to those of 8. Enzymatic
hydrolysis of 9 with naringinase gave the known coumarin derivative,
(3′S)-hydroxynodakenetin (Zhang and Yang, 2010) (9a) as the agly-
cone, and D-apiose and D-glucose. In the 1H and 13C NMR spectra of 9,
Fig. 3. Key HMBC correlations and ORTEP drawing of the crystal structure of 7.
3

Y. Matsuo, et al.
Phytochemistry173(2020)112301
compared, H-2″ and C-2″ of 14a were shifted downfield by 0.65 and
3.5 ppm and were observed at δH 4.46 and δC 81.6, respectively. Ac-
cordingly, the sulfate group was allocated to C-2″, and 14 was assigned
as 2″-sulfo-( )-oxypeucedanin hydrate.
3. Conclusions
In conclusion, a phytochemical examination of A. dahurica root gave
six benzofurans (1–6), a coumarin (7), and 14 furanocoumarins (8–21).
This is the first report of the isolation of naturally occurring fur-
anocoumarin sulfates, 14, 15, and 19, from A. dahurica, and of the
unique structure of 12, 13, and 18 because of the presence of a sucrose
group attached via an ether linkage. Compounds 1, 5, 6, 8, 9, and 14 at
sample concentration of 500 μM showed significant PPAR-γ ligand-
binding activity, among which the undescribed benzofuran derivate 1
showed the most potent activity. Compound 1 at 5.0 μM was shown to
induce adipocyte differentiation, involved in PPAR-γ ligand-binding
activity. Compound 1 is expected to be a possible lead compound for
anti-obesity agents.
Compound 15 (C16H16O9S) was considered to be a positional isomer
of 14 with regard to a sulfate group based on its 1H and 13C NMR
spectral data. Solvolysis of 15 in pyridine-dioxane gave 14a and an
aqueous solution containing sulfuric acid. When the 13C NMR spectrum
of 15 was compared with that of 14a, the C-3″ oxygenated quaternary
carbon signal at δC 72.7 in 14a was replaced by the downfield-shifted
carbon signal at δC 80.5 in 15. Compound 15 was thus characterized as
3″-sulfo-( )-oxypeucedanin hydrate. Although 14 and 15 were ob-
tained as optically active compounds, their corresponding desulfated
derivative (Fujioka et al., 1999) (14a) was a racemate; racemization
was presumed to have occurred during solvolysis. The absolute con-
figurations of 14 and 15 have yet to be determined.
4. Experimental
Compound 18 had the molecular formula of C29H38O17. Its 1H and
13C NMR spectral features indicated that 18 was related to 16 and 17,
and it was found that they shared the same furanocoumarin structure.
Acid hydrolysis of 18 yielded R-(+)-byakangelicin (Fujioka et al.,
1999) (18a), D-glucose, and D-fructose. The 1H and 13C NMR spectra of
18 exhibited signals for a sucrose unit, as observed for 12 and 13, and
HMBC correlations were observed from Glc-H2-6 at δH 3.74 (m) and
3.63 (dd, J = 10.3, 5.1 Hz) to C-3″ of the furanocoumarin moiety. Thus,
18 was identified as R-(+)-byakangelicin sucrose ether, as shown in
Fig. 1.
4.1. General experimental procedures
The instruments and experimental conditions, except for those
mentioned below, were the same as those described in a previous paper
(Matsuo et al., 2015). The melting point was recorded on an MP-3
melting apparatus (Yanaco, Kyoto, Japan). X-ray diffraction experi-
ments were conducted on a digital image projection (DIP) image plate
diffractometer (Bruker, AXS, Karlsruhe, Germany).
Compound 19 (C17H18O10S) was considered to be a sulfate of by-
akangelicin based on the molecular formula, IR spectrum with ab-
sorption bands at 1349 and 1072 cm−1, and 1H and 13C NMR spectra.
Solvolysis of 19 resulted in ( )-byakangelicin (Fujioka et al., 1999)
(19a) and sulfuric acid. When the 1H and 13C NMR spectra of 19 were
compared with those of 19a, H-2″ and C-2″ of 19a were observed to
have shifted downfield by 0.6 and 2.9 ppm and were observed at δH
4.43 and δC 81.2, respectively. Accordingly, the sulfate group was al-
located to C-2″, and 19 was identified as 2″-sulfo-( )-byakangelicin.
Compound 21 had the molecular formula C27H36O14, as revealed by
HR-ESI-TOF MS (m/z: 607.2004 [M + Na]+) and 13C NMR data. The
molecular formula was larger than that of 20 by C6H10O5, corre-
sponding to one hexosyl unit. Linkage of a β-D-glucopyranosyl group
(Glc) to the C-3″ in 21 was verified by a long-range correlation between
the anomeric proton of Glc at δH 4.55 (d, J = 7.8 Hz) and C-3″ of the
aglycone at δC 80.1 in the HMBC spectrum of 21. Thus, 21 was char-
acterized as 3″-O-β-D-glucopyranosyl-5,8-bis(2,3-dihydroxy-3-methyl-
butyloxy)-psoralen. The absolute configurations at C-2″ and C-2‴ re-
main to be determined.
4.2. Plant material
The dried root of Angelica dahurica (Hoffm.) Benth. & Hook.f. ex
Franch. & Sav. (Apiaceae) was obtained from Uchida Wakan-Yaku Co.,
Ltd. (Tokyo, Japan). The plant material was collected in Korea in 2012
(Lot. B320509). A voucher specimen was deposited at the Herbarium of
the Tokyo University of Pharmacy and Life Sciences (KS-2012-005).
4.3. Extraction and isolation
The dried roots of A. dahurica (10.5 kg) were extracted twice with
MeOH (40 L) at 50 °C for 2 h. The MeOH extract (1170 g) was passed
through a Diaion® HP-20 (Mitsubishi Chemical, Tokyo, Japan) column
(2000 g, 8.5 cm internal diameter (i.d.) × 60 cm). The 50% MeOH
(aq.)-eluted portion (155 g) was subjected to silica gel CC (1500 g,
8.0 cm i.d. × 40 cm) using CHCl3–MeOH–H2O (50:10:1) initially, and
finally MeOH, which provided 10 fractions (Frs. MH-1 to MH-10). Fr.
MH-3 was chromatographed on an ODS column (1000 g, 7.0 cm
i.d. × 30 cm) eluted with MeOH–H2O (2:3) to give 8 subfractions (Frs.
MH-3-1 to MH-3-8). Fr. MH-3-5 was subjected to silica gel CC (1200 g,
6.0 cm i.d. × 40 cm), eluted with CHCl3–MeOH–H2O (100:10:1;
40:10:1; 7:4:1) and ODS silica gel CC (1000 g, 7.0 cm i.d. × 29 cm)
eluted with MeCN–H2O (1:3) to yield 1 (4.5 mg), 7 (17.2 mg), and 11
(8.5 mg). Fr. MH-3-7 was subjected to ODS silica gel CC (1000 g, 7.0 cm
i.d. × 30 cm) eluted with MeOH–H2O (1:2; 1:1) to yield 2 (69.0 mg), 3
(4.5 mg), 4 (90.2 mg), 5 (26.3 mg), 6 (15.9 mg), and 9 (13.9 mg). The
MeOH eluted fraction (335 g) was chromatographed on silica gel
(1800 g, 8.0 cm i.d. × 36 cm), eluted with CHCl3–MeOH–H2O (7:4:1)
and finally with MeOH to provide 7 fractions (Frs. M-1 to M-7). Fr. M-3
was separated on a silica gel column (1200 g, 8.0 cm i.d. × 22 cm)
eluted with CHCl3–MeOH–H2O (50:10:1; 40:10:1; 20:10:1; 7:4:1) and
an ODS column (990 g, 6.0 cm i.d. × 30 cm) eluted with MeOH–H2O
(2:3) and MeCN–H2O (2:7) to yield 8 (5.4 mg), 10 (8.4 mg), 15
(9.0 mg), 16 (17.2 mg), 17 (3.7 mg), 19 (7.8 mg), and 21 (1.6 mg). Fr.
M-4 (487 mg) was subjected to CC on silica gel (1200 g, 8.0 cm
i.d. × 20 cm) eluted with CHCl3–MeOH–H2O (7:4:1) and ODS column
(700 g, 6.0 cm i.d. × 27 cm) eluted with MeCN–H2O (1:4; 1:3; 1:2) to
give 12 (13.5 mg), 13 (13.6 mg), 14 (28.1 mg), 18 (22.8 mg), and 20
(5.2 mg).
PPAR-γ is a ligand-dependent nuclear receptor expressed in adipose
tissue, liver, lung, kidney, and macrophages; it plays a major role in
lipid and glucose metabolism. Some coumarins obtained from A. da-
hurica root have been identified as PPAR-γ ligands (Han et al., 2018; Xi
et al., 2016). Thus, we evaluated PPAR-γ ligand-binding activity of the
isolated compounds at 500 and 50 μM using a nuclear receptor cofactor
assay system. Compounds 1, 5, 6, 8, 9, and 14 at 500 μM exhibited
significant PPAR-γ ligand-binding activity, among which the un-
described benzofuran derivative 1 showed the most potent activity
(Fig. 4). Glucosylation at the C-9 hydroxy group reduced PPAR-γ li-
gand-binding activity. Of the compounds with the furanocoumarin
skeleton (8–21), dehydrofuranocoumarin glycosides (8–11), and sul-
fated furanocoumarins (14, 15, 19) showed moderate PPAR-γ ligand-
binding activity. Next, we investigated the potential effect of 1 on the
differentiation of preadipocyte 3T3-L1 cells by Oil Red O staining
method. Compound 1 at 5.0 μM accumulated intracellular lipid in the
preadipocyte 3T3-L1 cells compared to the control (Fig. 5). These re-
sults indicate that 1 shows potent PPAR-γ ligand-binding activity in
3T3-L1 cells, leading to adipocyte differentiation.
4

Y. Matsuo, et al.
Phytochemistry173(2020)112301
Fig. 4. PPAR-γ ligand binding activity of 1–21. Pioglitazone (PG): 50 (■) and 5.0 (□) μM, 1–21: 500 and 50 μM. Data are represented as the mean
Dunnett's test was used for statistical analysis. ***p < 0.001, **p < 0.01, *p < 0.05.
S.E.M. (n = 3).
V = 661.73 (3)Å3, Z = 2; T = 296 K, dcalc = 1.407 Mg/m3; μ (Mo Kα,
λ = 0.71073 Å) = 0.11 mm−1; R(all) = 0.0409, R(gt) = 0.0370, wR
(ref) = 0.1081, wR(gt) = 0.1054. Crystallographic data of 7 have been
deposited with the Cambridge Crystallographic Data Centre (CCDC) as
supplementary publication no. CCDC 1903140. Copies of the data can
be obtained free of charge from the CCDC via http://beta-www.ccdc.
cam.ac.uk/pages/Home.aspx.
4.3.1. Structural characterization
3-[6,7-furano-9-hydroxy-4-(2″,3″-dihydroxy-3″-methylbutyloxy)]-
phenyl propionic acid (1): Amorphous solid; C16H20O7; [α]D25 +26.0 (c
0.94, MeOH); HR-ESI-TOF-MS m/z: 347.1107 [M + Na]+ (calcd for
C
16H20NaO7: 347.1107); IR νmax (film) cm−1: 3362 (OH), 1714 (C]O),
1558 and 1455 (aromatic ring); UV λmax (MeOH) nm (log ε): 283
(3.55), 255 (3.89), 241 (3.81); 1H and 13C NMR (500 and 125 MHz,
CD3OD): see Table 1.
(3′S)-hydroxy-nodakenetin 4′-O-β-D-apiofuranosyl-(1
→ 6)-β-D-
glucopyranoside (9): Amorphous solid; C25H32O14; [α]D25 -26.2 (c 0.10,
3-[6,7-furano-9-(β-D-glucopyranosyloxy)-4-(2″,3″-dihydroxy-3″-
MeOH); HR-ESI-TOF-MS m/z: 579.1687 [M
+
Na]+ (calcd. for
methylbutyloxy)]-phenyl propionic acid (2): Amorphous solid;
C25H32NaO14: 579.1690); IR νmax (film) cm−1: 3385 (OH), 1719 (C]
O), 1626 and 1572 (aromatic ring); UV λmax (MeOH) nm (log ε): 326
(3.37), 266 (3.09), 236 (3.25), 226 (3.46); 1H and 13C NMR (500 and
125 MHz, CD3OD): see Table 1.
25
C
22H30O12
;
[α]D
-29.0 (c 0.10, MeOH); HR-ESI-TOF-MS m/z:
509.1627 [M + Na]+ (calcd for C22H30NaO12: 509.1635); IR νmax
(film) cm−1: 3364 (OH), 1716 (C]O), 1557 and 1455 (aromatic ring);
UV λmax (MeOH) nm (log ε): 286 (3.24), 252 (3.93), 236 (3.74); 1H and
13C NMR (500 and 125 MHz, CD3OD): see Table 1.
25
Compound 9a: (3′S)-hydroxynodakenetin; C14H14O5; [α]D -22.5
(c 0.05, CHCl3); HR-ESI-TOF-MS m/z: 285.0733 [M + Na]+ (calcd for
3-[6,7-furano-9-(β-D-glucopyranosyloxy)-4-(2″,3″-dihydroxy-3″-
methylbutyloxy)]-phenyl propionic acid methyl ester (3): Amorphous
C
14H14NaO5: 285.0739); 13C NMR (125 MHz, pyridine) δ: 163.2, 112.8,
25
146.1, 126.5, 129.8, 164.6, 99.1, 158.0, 114.7 (C-2-C-10), 93.0, 72.1,
73.1, 27.0, 26.4 (C-2′-C-6′).
solid; C23H32O12; [α]D -10.0 (c 0.15, MeOH); HR-ESI-TOF-MS m/z:
523.1790 [M + Na]+ (calcd for C23H32NaO12: 523.1791); IR νmax
(film) cm−1: 3419 (OH), 1730 (C]O), 1615 and 1592 (aromatic ring);
UV λmax (MeOH) nm (log ε): 279 (3.33), 252 (3.82), 237 (3.67). 1H and
13C NMR (500 and 125 MHz, CD3OD): see Table 1.
R-(+)-oxypeucedanin hydrate sucrose ether (12): Amorphous solid;
25
C
28H36O16; [α]D +29.4 (c = 0.10, MeOH); HR-ESI-TOF-MS m/z:
651.1897 [M + Na]+ (calcd. for C28H36NaO16: 651.1901); IR νmax
(film) cm−1: 3214 (OH), 1699 (C]O), 1559 and 1455 (aromatic ring);
UV λmax (MeOH) nm (log ε): 312 (3.63), 250 (3.79), 221 (3.98). 1H and
13C NMR (500 and 125 MHz, CD3OD): see Table 1.
7-hydroxy-6-(2′,3′,4′-trihydroxy-isopentanyl)-coumarin (7): Triclinic
crystal; C14H16O6; mp: 199–203 °C; [α]D25 +0.3 (c 0.10, MeOH); HR-ESI-
TOF-MS m/z: 303.0838 [M + Na]+ (calcd for C14H16NaO6: 303.0845);
IR νmax (film) cm−1: 3391 (OH), 1714 (C]O), 1683 and 1620 (aromatic
ring); UV λmax (MeOH) nm (log ε): 332 (4.53), 266 (3.67), 224 (4.54); 1H
and 13C NMR (500 and 125 MHz, CD3OD): see Table 1.
25
Compound 12a: R-(+)-oxypeucedanin hydrate; C16H16O6; [α]D
+14.7 (c = 0.10, MeOH); HR-ESI-TOF-MS m/z: 327.0849 [M + Na]+
(calcd for C16H16NaO6: 327.0845).
13C NMR (125 MHz, CD3OD) δ: 163.3, 113.0, 141.7, 150.8, 115.4,
159.9, 94.7, 153.9, 108.4 (C-2-C-10), 146.8, 106.2 (C-2′, C-3’), 75.8,
X-Ray Crystallography of 7: Triclinic, space group P-1, unit cell
dimension a = 7.1576 (8)Å, b = 8.0135 (9)Å, c = 11.8273 (13),
Fig. 5. Effect of 1 on lipid accumulation in 3T3-L1 adipocytes. Subconfluent 3T3-L1 preadipocytes in the basal medium were treated in the presence or absence of
5 μM of 1 for 96 h 3T3-L1 cells treated in the differentiation-inducing medium are shown as a positive control. Intracellular lipid was stained with Oil Red O. (For
interpretation of the references to colour in this figure legend, the reader is referred to the Web version of this article.)
5

Y. Matsuo, et al.
Phytochemistry173(2020)112301
Table 1
1H and 13C NMR (500 and 125 MHz, CD3OD) spectral assignments for 1–3, 7, 9, 12–15, 18–19, and 21.
1
2
Position
δH
J (Hz)
δC
Position
δH
J (Hz)
δC
1
2
–
182.9
39.2
1
2
–
182.1
38.7
2.51 (2H)
t
6.9
2.43 (2H)
3.13 (2H)
–
m
3
2.98 (2H)
dt
14.0, 6.9
21.5
3
t-like
8.1
22.0
4
–
117.8
151.8
112.6
157.1
95.0
4
118.6
151.5
114.6
156.6
94.7
5
–
5
–
6
–
6
–
7
–
7
–
8
6.64
–
d
0.8
8
7.09
–
s
9
155.5
143.3
105.6
75.2
9
155.8
144.4
105.6
75.5
2′
3′
1″a
1″b
2″
3″
4″
5″
7.46
6.90
4.47
4.16
3.80
–
d
2.2
2′
3′
1″
7.57
6.96
4.5
d
2.5
dd
dd
dd
dd
2.2, 0.8
9.8, 2.5
9.8, 8.3
8.3, 2.5
d
2.5
dd
dd
dd
9.0, 2.5
9.0, 8.5
8.5, 2.5
4.17
3.82
–
78.5
72.7
26.8
25.0
2″
3″
4″
5″
Glc-1
2
78.4
72.7
27.0
24.8
103.4
74.8
77.9
71.4
78.1
62.6
1.28
1.22
s
s
1.28
1.22
4.89
3.57
3.48
4.42
3.48
3.92
3.72
s
s
d
8.5
dd
dd
dd
m
dd
dd
8.5, 8.0
9.0, 8.5
9.5, 9.0
3
4
5
6a
6 b
12.0, 2.0
12.0, 8.3
3
7
Position
δH
J (Hz)
δC
Position
δH
J (Hz)
δC
1
2
–
176.6
2
3
–
163.9
112.1
146.2
131.6
126.6
161.5
103.1
155.7
112.9
32.8
2.61 (2H)
3.14 (2H)
–
m
35.2
20.6
6.16
7.83
7.40
–
d
d
s
9.4
9.4
3
m
4
4
117.5
151.6
114.8
156.9
95.1
5
5
–
6
6
–
7
–
7
–
8
6.72
–
s
8
7.11
–
s
9
9
155.6
144.7
105.6
75.4
10
1′a
1′b
2′
3′
4′
–
2′
3′
1″
7.59
6.98
4.47
4.16
3.78
–
d
2.2
3.12
2.64
3.82
–
dd
dd
dd
14.1, 2.3
14.1, 10.5
10.5, 2.3
d
2.2
dd
dd
dd
9.8, 2.6
9.8, 8.2
8.2, 2.6
76.1
75.5
68.6
2″
3″
78.5
72.7
26.8
25.0
103.2
75.0
78.3
71.4
78.2
62.6
3.62
3.52
1.20
d
d
s
10.8
10.8
4″
1.27
1.22
4.92
3.51
3.48
3.4
s
5′
19.1
5″
s
Glc-1
2
d
7.2
dd
dd
dd
m
dd
dd
s
8.5, 7.2
8.9, 8.5
9.1, 8.9
3
4
5
3.46
3.91
3.71
3.65
6a
6 b
OMe
12.0, 2.2
12.0, 5.6
52.1
(continued on next page)
6

Y. Matsuo, et al.
Phytochemistry173(2020)112301
Table 1 (continued)
9
12
Position
δH
J (Hz)
δC
Position
δH
J (Hz)
δC
2
3
–
160.9
112.3
144.3
126.0
129.2
163.0
98.1
2
3
–
163.4
112.7
142.0
151.0
114.8
159.9
94.3
6.26
7.64
7.61
–
d
d
s
9.5
9.5
6.28
8.47
–
d
d
9.8
9.8
4
4
5
5
6
6
–
7
–
7
–
8
6.83
–
s
8
7.17
–
s
9
156.9
113.4
92.7
9
153.9
107.9
146.7
106.7
75.3
10
2′
3′
4′
5′
6′
Glc-1
2
–
10
2′
–
4.7
5.62
–
d
d
6.5
6.5
7.79
7.26
4.95
4.40
4.13
–
d
2.4
71.4
3′
d
2.4
78.6
1″a
1″b
2″
3″
4″
5″
Glc-1
2
dd
dd
dd
9.7, 2.3
9.7, 9.0
9.0, 2.3
1.9
1.86
5.28
3.87
4.18
3.99
3.97
4.29
4.03
5.64
4.66
–
s
25.9
s
23.2
75.8
77.9
23.2
21.7
93.1
73.2
74.8
71.7
74.4
62.8
d
7.5
98.7
t-like
dd
dd
m
8.5, 7.5
8.5, 7.6
7.6, 7.6
74.8
1.35
1.25
5.19
3.09
3.62
3.15
3.78
3.66
3.82
3.50
3.46
–
s
3
78.5
s
4
71.5
d
3.9
5
76.6
dd
dd
dd
ddd
dd
dd
d
9.3, 3.9
9.3, 9.1
9.7, 9.1
9.7, 5.3, 2.0
11.3, 2.0
11.3, 5.3
12.3
6a
6 b
Api-1
2
brd
dd
d
10.5
68.1
3
10.5, 5.8
2.5
4
110.9
77.8
80.4
75.0
5
d
2.5
6a
6 b
Fru-1a
1 b
2
3
4
4.55
4.34
4.15
d
d
s
9.5
9.5
64.0
d
12.3
5(2H)
65.6
105.1
79.2
76.7
82.5
65.2
3
4.04
3.94
3.82
3.90
3.68
d
8.4
4
dd
m
8.4, 8.4
5
6a
6 b
dd
dd
9.9, 7.9
7.9, 2.4
13
14
Position
δH
J (Hz)
δC
Position
δH
J (Hz)
δC
2
3
–
163.3
2
3
–
163.6
112.7
142.7
150.8
114.8
159.9
94.2
6.29
8.43
–
d
9.8
9.8
112.9
141.8
150.8
115.5
159.8
94.6
6.28
8.62
–
d
d
9.7
9.7
4
d
4
5
5
6
–
6
–
7
–
7
–
8
7.17
–
s
8
7.16
–
s
9
153.9
108.4
146.8
106.3
75.9
9
154.0
108.1
146.6
106.6
74.4
10
2′
–
10
2′
–
7.79
7.24
4.82
4.44
3.96
–
d
2.4
7.77
7.29
4.79
4.67
4.46
–
d
2.3
3′
d
2.4
3′
d
2.3
1″a
1″b
2″
3″
4″
5″
Glc-1
2
dd
dd
dd
10.1, 2.3
10.1, 8.3
8.3, 2.3
1″a
1″ b
2″
3″
4″
5″
dd
dd
ddd
9.6, 4.0
9.6, 5.4
9.8, 5.4, 4.0
78.1
79.2
23.8
24.5
94.1
72.9
75.9
71.3
74.6
62.3
81.6
73.2
26.6
26.4
1.37
1.34
5.40
3.39
3.97
3.37
3.85
3.79
3.72
3.67
3.61
–
s
1.38
1.35
s
s
s
d
3.6
dd
dd
dd
ddd
dd
dd
d
9.5, 3.6
9.5, 8.9
10.0, 8.9
10.0, 4.4, 2.4
11.8, 2.4
11.8, 4.4
12.3
3
4
5
6a
6 b
Fru-1a
1 b
2
64.3
d
12.3
105.3
79.7
75.8
83.8
63.4
3
4.09
4.04
3.77
3.76
3.76
d
8.1
4
dd
m
m
m
8.1, 7.6
5
6a
6 b
(continued on next page)
7

Y. Matsuo, et al.
Phytochemistry173(2020)112301
Table 1 (continued)
15
18
Position
δH
J (Hz)
δC
Position
δH
J (Hz)
δC
2
3
–
163.4
112.8
142.0
151.0
115.4
159.9
94.4
2
3
–
162.8
113.2
141.5
146.1
116.4
151.7
128.5
144.9
108.7
147.1
106.3
6.28
8.45
–
d
d
9.8
9.8
6.30
8.26
–
d
d
9.8
9.8
4
4
5
5
6
–
6
–
7
–
7
–
8
7.18
–
s
8
–
9
153.9
108.3
146.7
106.7
76.0
9
–
10
2′
3′
1″a
1″b
2″
3″
4″
5″
–
10
2′
–
7.77
7.34
4.91
4.51
3.98
–
d
2.4
7.87
7.22
4.60
4.32
3.99
–
d
2.3
2.3
d
2.4
3′
d
dd
dd
dd
10.0, 2.6
10.0, 8.2
8.2, 2.6
1″a
1″b
2″
3″
4″
5″
OMe
Glc-1
2
dd
dd
dd
10.4, 2.6
10.4, 8.1
8.1, 2.6
76.6
78.5
80.5
24.3
26.3
77.1
78.0
22.6
21.6
61.5
93.5
73.2
74.5
71.7
73.5
62.3
1.54
1.51
s
s
1.27
1.25
4.21
5.31
3.37
3.68
3.34
3.88
3.74
3.63
3.59
3.56
–
s
s
s
d
3.8
dd
dd
dd
ddd
m
dd
d
9.9, 3.8
9.9, 9.6
10.0, 9.6
10.0, 5.1, 1.9
3
4
5
6a
6 b
Fru-1a
1 b
2
10.3, 5.1
12.3
64.2
d
12.3
105.3
79.4
75.6
83.8
63.2
3
4.07
4.04
3.73
3.72
d
8.2
4
dd
m
m
8.2, 7.7
5
6 (2H)
19
δH
–
21
Position
J (Hz)
δC
Position
δH
J (Hz)
δC
2
3
162.5
113.2
141.4
146.0
116.4
151.4
128.3
144.6
108.7
147.0
106.3
75.6
2
3
–
162.7
113.2
142.0
145.4
117.8
151.4
128.9
144.8
109.8
147.1
106.4
76.6
6.28
d
9.8
9.8
6.32
8.43
–
d
d
9.8
9.8
4
8.24
–
d
4
5
5
6
–
6
–
7
–
7
–
8
–
8
–
9
–
9
–
10
2′
–
10
2′
–
7.83
7.22
4.59
4.54
4.43
–
d
2.3
7.84
7.24
4.75
4.28
3.97
–
d
2.3
3′
d
2.3
3′
d
2.3
1″a
1″b
2″
3″
4″
5″
OMe
dd
dd
ddd
10.1, 5.9
10.1, 4.2
9.9, 5.9, 4.2
1″a
1″b
2″
3″
4″
5″
1‴a
1‴b
2‴
3‴
4‴
5‴
Glc-1
2
dd
dd
dd
10.1, 2.2
10.1, 8.3
8.3, 2.2
81.2
73.7
26.8
25.6
61.5
77.4
80.1
24.3
22.4
76.8
1.43
1.41
4.21
s
s
s
1.36
1.32
4.59
4.31
3.84
–
s
s
dd
dd
dd
10.2, 2.8
10.2, 8.1
8.1, 2.8
78.3
72.7
26.7
25.0
98.5
75.3
78.2
71.7
77.8
62.8
1.27
1.22
4.55
3.16
3.37
3.26
3.27
3.81
3.62
s
s
d
7.8
dd
dd
dd
m
dd
dd
9.0, 7.8
9.0, 8.9
8.9, 7.3
3
4
5
6a
6 b
11.4, 1.8
11.4, 6.1
78.1, 72.7, 24.8, 27.2 (C-1″-C-5″).
(C]O), 1648 and 1457 (aromatic ring); UV λmax (MeOH) nm (log ε):
311 (4.05), 249 (4.22), 220 (4.40). 1H and 13C NMR (500 and 125 MHz,
CD3OD): see Table 1.
25
Compound 13: Amorphous solid;
C
28H36O16
;
[α]D
-192.9
(c = 0.10, MeOH); HR-ESI-TOF-MS m/z: 651.1903 [M + Na]+ (calcd.
for C28H36NaO16: 651.1901); IR νmax (film) cm−1: 3394 (OH), 1736
2″-sulfo-( )-oxypeucedanin hydrate (14): Amorphous solid;
8

Y. Matsuo, et al.
Phytochemistry173(2020)112301
25
C16H16O9S; [α]D
-4.1 (c = 0.10, MeOH); HR-ESI-TOF-MS m/z:
(dioxane/H2O, 1:1, 2 mL) and heated at 90 °C for 1 h. After cooling, the
reaction mixture was neutralized by passage through an Amberlite IRA-
96 S column (Organo, Tokyo, Japan) and then passed through a Diaion®
HP-20 (16 mm i.d. × 200 mm) eluted with MeOH–H2O (2:5), acetone-
EtOH (1:1), and finally with MeOH to yield 12a (1.9 mg from 12;
1.7 mg from 13) and 18a (2.4 mg), and sugar fractions. HPLC analysis
of the sugar fractions under the same conditions as in the case of 2
showed the presence of D-glucose (positive optical rotation, tR 11.97,
12.52, and 12.38 min) and D-fructose (negative optical rotation, tR
9.26, 9.72, and 9.78 min).
407.0408 [M + Na]+ (calcd for C16H16NaO9S: 407.0413); IR νmax
(film) cm−1: 3360 (OH), 2921 and 2852 (CH), 1726 (C]O), 1622 and
1457 (aromatic ring), 1349 and 1097 (S]O); UV λmax (MeOH) nm
(log ε): 310 (3.93), 268 (4.03), 249 (4.06), 222 (4.21); 1H and 13C NMR
(500 and 125 MHz, CD3OD): see Table 1.
25
Compound 14a: ( )-oxypeucedanin hydrate; C16H16O6; [α]D
-1.2 (c = 0.06, MeOH); HR-ESI-TOF-MS m/z: 327.0845 [M + Na]+
(calcd. for C16H16NaO6: 327.0845).
3″-sulfo-( )-oxypeucedanin hydrate (15): Amorphous solid;
25
C
16H16O9S; [α]D +7.3 (c = 0.08, MeOH); HR-ESI-TOF-MS m/z:
407.0418 [M + Na]+ (calcd. for C16H16NaO9S: 407.0413); IR νmax
(film) cm−1: 3360 (OH), 2920 and 2851 (CH), 1720 (C]O), 1623 and
1458 (aromatic ring), 1349 and 1075 (S]O); UV λmax (MeOH) nm
(log ε): 311 (3.82), 267 (3.89), 250 (3.94), 221 (4.10); 1H and 13C NMR
(500 and 125 MHz, CD3OD): see Table 1.
4.3.3. Solvolysis
Solvolysis of 14, 15, and 19: Compounds 14 (8.0 mg), 15 (7.5 mg),
and 19 (5.0 mg) were independently treated with pyridine-dioxane
(1:1, 2 mL) at 100 °C for 3 h. The crude reaction mixture was neu-
tralized with 5% NaHCO3 (aq) and extracted with EtOAc (10 mL × 3).
The EtOAc extract was purified by silica gel CC to yield 14a (3.5 mg
from 14; 3.0 mg from 15) and 19a (1.9 mg). The aqueous solutions
were independently concentrated to approximately 2 mL, and an aqu-
eous solution of 0.1 M BaCl2 was added. The white precipitate formed
and collected by centrifugation was not soluble on the addition of 2 M
HCl.
R-(+)-byakangelicin sucrose ether (18): Amorphous solid;
25
C29H38O17; [α]D +29.4 (c = 0.10, MeOH); HR-ESI-TOF-MS m/z:
681.2009 [M + Na]+ (calcd. for C29H38NaO17: 681.2007); IR νmax
(film) cm−1: 3402 (OH), 1734 (C]O), 1591 and 1464 (aromatic ring);
UV λmax (MeOH) nm (log ε): 313 (3.52), 268 (3.73), 222 (3.96); 1H and
13C NMR (500 and 125 MHz, CD3OD): see Table 1.
25
Compound 18a: (+)-byakangelicin; C17H18O7; [α]D +8.8 (c =
0.10, MeOH); HR-ESI-TOF-MS m/z: 357.0951 [M + Na]+ (calcd. for
C
17H18NaO7: 357.0950). 13C NMR (125 MHz, CD3OD) δ: 162.7, 113.2,
4.4. PPAR-γ ligand binding activity
141.5, 145.7, 116.4, 151.7, 128.5, 145.0, 108.7 (C-2-C-10), 147.0,
106.4 (C-2′, C-3′), 76.9, 78.3, 72.7, 26.7, 25.1 (C-1″-C-5″), 61.5 (OMe).
2″-sulfo-( )-byakangelicin (19): Amorphous solid; C17H18O10S;
PPAR-γ agonistic activity was examined using a nuclear receptor
cofactor assay system (EnBio RCAS for PPAR-γ, EnBioTec Laboratories,
Tokyo, Japan) according to the manufacturer's instructions. Briefly, a
cyclic AMP response element binding protein (CBP)-derived peptide
was immobilized on the bottom of a microtiter plate. After the addition
of recombinant human PPAR-γ solution, dimethyl sulfoxide (DMSO) as
a control, positive control at 50 or 5.0 μM, or isolated compounds at
500 or 50 μM were added to respective wells. Binding of PPAR-γ ligand
complex to the CBP on the plate was detected by measuring the ab-
sorbance at 450 nm. This assay involves a cell-free system using nuclear
receptors and cofactors. Pioglitazone (Sigma-Aldrich, St. Louis, MO,
USA) was used as a positive control.
25
[α]D -1.7 (c = 0.10, MeOH); HR-ESI-TOF-MS m/z: 437.0522 [M +
Na]+ (calcd. for C17H18NaO10S: 437.0518); IR νmax (film) cm−1: 3360
(OH), 1721 (C]O), 1622 and 1458 (aromatic ring), 1349 and 1072
(S]O); UV λmax (MeOH) nm (log ε): 312 (3.84), 269 (4.03), 241 (3.94),
223 (4.20); 1H and 13C NMR (500 and 125 MHz, CD3OD): see Table 1.
25
Compound 19a: ( )-byakangelicin; C17H18O7; [α]D +0.6 (c =
0.10, MeOH); HR-ESI-TOF-MS m/z: 357.0950 [M + Na]+ (calcd for
C
17H18NaO7: 357.0950).
3″-O-β-D-glucopyranosyl-5,8-bis(2,3-dihydroxy-3-methylbutyloxy)-
25
psoralen (21): Amorphous solid; C27H36O14; [α]D -1.7 (c = 0.08,
MeOH); HR-ESI-TOF-MS m/z: 607.2004 [M
+
Na]+ (calcd. for
C
27H36NaO14: 607.2003); IR νmax (film) cm−1: 3392 (OH), 1736 (C]
4.5. Cell culture
O), 1603 and 1462 (aromatic ring); UV λmax (MeOH) nm (log ε): 311
(3.90), 268 (4.09), 249 (4.03); 1H and 13C NMR (500 and 125 MHz,
CD3OD): see Table 1.
The mouse fibroblast 3T3-L1 cell line (ATCC CL-173) was cultured
with basal medium containing fetal bovine serum and antibiotics
(Omental Adipocyte Differentiation Medium, Zen-Bio, NC, USA). After
the cells were grown to sub-confluency, cell differentiation was induced
by culturing in the differentiation-inducing medium with 3-isobutyl-1-
methylxanthine and PPAR-γ agonist (diluted two-fold in Omental
Adipocyte Differentiation Medium, Zen-Bio, Research Triangle Park,
NC, USA) for 96 h.
4.3.2. Hydrolysis
Enzymatic hydrolysis of 2: Compound 2 (15.0 mg) was treated with
β-D-glucosidase (EC232-589-7, 200 mg) in AcOH/AcONa buffer (pH
5.0, 15.0 mL) for 24 h. The crude hydrolysate was chromatographed on
silica gel (6 mm i.d. × 200 mm) eluted with CHCl3–MeOH–H2O
(20:10:1) to yield 1 (12.0 mg) and a sugar fraction. The sugar fraction
was analyzed by HPLC under the following conditions: Capcell Pak NH2
UG80 (4.6 mm i.d. × 250 mm, 5 μm, Shiseido, Tokyo, Japan); mobile
phase of MeCN–H2O (7:3); detection by refractive index and optical
rotation; and flow rate of 1.0 mL/min. D-Glucose was identified by
comparing its retention time and optical rotation with those of an au-
thentic sample. tR (min): 14.70 (D-glucose, positive optical rotation).
Enzymatic hydrolysis of 9: Compound 9 (11.0 mg) was treated with
naringinase (EC 232-96-4, 400 mg) in AcOH/AcOK buffer (pH 4.3,
4.0 mL) for 24 h. The crude hydrolysate was chromatographed on silica
gel (16 mm i.d. × 200 mm) eluted with CHCl3–MeOH (3:1) and finally
with MeOH to yield 9a (0.9 mg) and a sugar fraction. HPLC analysis of
the sugar fraction under the same conditions as in the case of 2 showed
the presence of D-glucose (positive optical rotation, tR 15.05 min) and
D-apiose (positive optical rotation, tR 7.17 min).
4.6. Oil red O staining
Oil Red O staining of the cells was performed using a standard
protocol. Briefly, after fixation with 4% paraformaldehyde for 10 min,
the cells were incubated with 3 mg/mL Oil Red O (Sigma-Aldrich, St.
Louis, MO, USA) in 60% isopropanol for 30 min. After removing the
solution, the cells were washed with 60% isopropanol and subsequently
with PBS. Oil red staining was evaluated using total photographic
images of the staining, which were divided into multiple fields.
Funding
Acid hydrolysis of 12, 13, and 18: Compounds 12 (4.8 mg), 13
(5.5 mg), and 18 (5.3 mg) were independently dissolved in 1 M HCl
This work was supported by JSPS KAKENHI Grant Number
JP17K08346.
9

Y. Matsuo, et al.
Phytochemistry173(2020)112301
Acknowledgments
Kwon, Y.S., Kobayashi, A., Kajiyama, S.I., Kawazu, K., Kanzaki, H., Kim, C.M., 1997.
Antimicrobial constituents of Angelica dahurica roots. Phytochemistry 44, 887–889.
Liu, D.P., Luo, Q., Wang, G.H., Xu, Y., Zhang, X.K., Chen, Q.C., Chen, H.F., 2011.
Furocoumarin derivatives from radix Angelicae dahuricae and their effects on RXRα
transcriptional regulation. Molecules 16, 6339–6348.
We thank Kazuhiro Tamura and Kazuya Kusama for the 3T3-L1 cells
assays and helpful advice.
Marumoto, S., Miyazawa, M., 2010. β-secretase inhibitory effects of furanocoumarins
from the root of Angelica dahurica. Phytother Res. 24, 510–513.
Appendix A. Supplementary data
Matsuo, Y., Takaku, R., Mimaki, Y., 2015. Novel steroidal glycosides from the bulbs of
Lilium pumilum. Molecules 20, 16255–16265.
Niu, X.M., Li, S.H., Jiang, B., Zhao, Q.S., Sun, H.D., 2002. Constituents from the roots of
Heracleum rapula franch. J. Asian Nat. Prod. Res. 4, 33–41.
Supplementary data to this article can be found online at https://
doi.org/10.1016/j.phytochem.2020.112301.
Oh, H., Lee, H.S., Kim, T., Chai, K.Y., Chung, H.T., Kwon, T.O., Yun, Y.G., 2002.
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