Structurally diverse glycoconjugated volatile compounds from Oxytropis falcata Bunge

Article abstract of DOI:10.1016/j.phytol.2018.07.015

A phytochemical investigation on the whole plant of Oxytropis falcata Bunge yielded 16 glycoconjugated compounds with structurally diverse volatile aglycones but similar sugars. Of these, five were previously undescribed compounds with different volatile aglycones but same glucuronic acid (1 → 2) glucosyl moiety, including three octanol glucuronic acid (1 → 2) glucosides (1–3), one acyclic monoterpenoid glucuronic acid (1 → 2) glucoside (4), and one 4-phenyl-butan-2-ol glucuronic acid (1 → 2) glucoside (5), and 11 were biological related known glucoconjugated volatile compounds (6–16) isolated from genus Oxytropis for the first time. The structures of these compounds were determined by extensive spectroscopic analysis of MS, 1D and 2D NMR data. The absolute configurations of aglycones and sugar residues were assigned via enzymatic hydrolysis and subsequently comparison of the specific rotations. This is the first report of such structurally diverse glycoconjugated volatile compounds from O. falcata, which might be regarded as the precursor of free volatile compounds, and presents scientific evidences for better clarifying the volatile compositions of this medicinal plant and genus Oxytropis.

Full text of DOI:10.1016/j.phytol.2018.07.015

PhytochemistryLetters27(2018)143–147
Contents lists available at ScienceDirect
Phytochemistry Letters
journal homepage: www.elsevier.com/locate/phytol
Structurally diverse glycoconjugated volatile compounds from Oxytropis
falcata Bunge
Shanshan Wanga, Xiaojing Zhanga, Sheng Queb, Hong Lianga, Pengfei Tua, Qingying Zhanga,⁎
a
State Key Laboratory of Natural and Biomimetic Drugs and Department of Natural Medicines, School of Pharmaceutical Sciences, Peking University Health Science
Center, Beijing 100191, People’s Republic of China
b
Department of Chemistry, Teachers College for Nationalities, Qinghai Normal University, Xining 810008, People’s Republic of China
A R T I C L E I N F O
A B S T R A C T
Keywords:
Oxytropis
Oxytropis falcata
Glycoconjugated volatile compounds
Volatile compositions
A phytochemical investigation on the whole plant of Oxytropis falcata Bunge yielded 16 glycoconjugated com-
pounds with structurally diverse volatile aglycones but similar sugars. Of these, five were previously undescribed
compounds with different volatile aglycones but same glucuronic acid (1 → 2) glucosyl moiety, including three
octanol glucuronic acid (1 → 2) glucosides (1–3), one acyclic monoterpenoid glucuronic acid (1 → 2) glucoside
(4), and one 4-phenyl-butan-2-ol glucuronic acid (1 → 2) glucoside (5), and 11 were biological related known
glucoconjugated volatile compounds (6–16) isolated from genus Oxytropis for the first time. The structures of
these compounds were determined by extensive spectroscopic analysis of MS, 1D and 2D NMR data. The ab-
solute configurations of aglycones and sugar residues were assigned via enzymatic hydrolysis and subsequently
comparison of the specific rotations. This is the first report of such structurally diverse glycoconjugated volatile
compounds from O. falcata, which might be regarded as the precursor of free volatile compounds, and presents
scientific evidences for better clarifying the volatile compositions of this medicinal plant and genus Oxytropis.
1. Introduction
falcata, and forty-four flavonoids, including five characteristic 3-hy-
droxy-3-methylglutaryl-rhamnocitrin 3-O-glycosides and three novel
Genus Oxytropis (Leguminosae), including 300 species, grows in the
high latitude zones of the world, especially in Central and East Asia,
Europe, Africa and North America. As an important part of grassland in
mountainous regions, some Oxytropis plants are toxic, and a few can be
used as forage. In China, certain Oxytropis species are used as folk
medicines in Tibetan or Mongolian medicines, such as O. falcata Bunge
and O. chiliophylla Royle, which are officially documented as the bo-
tanical origin of Tibetan medicine “Er-Da-Xia” in Chinese
Pharmacopoeia for the treatment of inflammation, pyreticsis and
bleeding for thousands of years (Diamer, 1986; Committee of Chinese
Pharmacopoeia, 2015). In the past decade, a number of phytochemical
investigations mainly focusing on the ethanol extract of O. falcata had
been carried out, showing that flavonoids (Chen et al., 2010a; Wang
et al., 2012; Zhang et al., 2013, 2014), oleanane-type triterpenoids
(Chen et al., 2009) and N-benzoylindole analogues (Chen et al., 2010b)
were the principle constituents, among which flavonoids exhibited
potent anti-inflammatory, analgesic and antiproliferative activities
(Yang et al., 2010; Chen et al., 2011). Our research group had carried
out a systematic investigation on the bioactive constituents of O.
chalcone dimers fused through a cyclobutane ring (Wang et al., 2012;
Zhang et al., 2013, 2014), had been isolated. As some Oxytropis species
that have stimulating odors, such as O. kansuensis Bunge being reported
to have strong stimulating effect on the human eyes and respiratory
system and cause eyes congestion, headache and throat discomfort
(Liang et al., 1994), O. falcata also releases specific odors and causes
headache and discomfort. However, studies on the volatile composi-
tions of O. falcata are quite few, and only one paper reported the
identification of 58 compounds, including paraffin hydrocarbons, fla-
vonoids, aldehydes, terpenoids, organic acids, esters, and nitrogenous
compounds, from the volatile oils of aerial parts of O. falcata extracted
by water distillation, supercritical CO2 and headspace extraction tech-
niques, using GC and GC/MS methods (Wang et al., 2010). As part of
our ongoing study on the bioactive constituents of O. falcata, five pre-
viously undescribed glucuronic acid (1 → 2) glucosidated compounds
with different volatile aglycones (1–5), together with eleven biological
related known glucoconjugated volatile compounds (6–16), were iso-
lated from O. falcata. These compounds might be regarded as the pre-
cursor of free volatile compounds, and presented scientific evidences
⁎
Corresponding author at: State Key Laboratory of Natural and Biomimetic Drugs and Department of Natural Medicines, School of Pharmaceutical Sciences, Peking
University Health Science Center, 38 Xueyuan Road, Beijing 100191, People’s Republic of China.
E-mail address: qyzhang@hsc.pku.edu.cn (Q. Zhang).
https://doi.org/10.1016/j.phytol.2018.07.015
Received 10 May 2018; Received in revised form 2 July 2018; Accepted 13 July 2018
Availableonline20July2018
1874-3900/©2018PhytochemicalSocietyofEurope.PublishedbyElsevierLtd.Allrightsreserved.

S. Wang et al.
PhytochemistryLetters27(2018)143–147
Fig. 1. Structures of compounds 1–5.
for better clarifying the volatile compositions of this medicinal plant
and genus Oxytropis. Herein, the isolation, structural elucidation, and
possible biogenetic significance of these compounds are described.
C-2′. 3R configuration was assigned from the optical rotation value of
aglycone ([α]3D0 = +6.7 (c 0.27, CH2Cl2)) that was obtained by enzy-
matic hydrolysis (Schwab et al., 1989). Thus, compound 2 was char-
acterized unambiguously as (3R)-octan-1, 3-diol 3-O-β-D-glucuronic
acid-(1 → 2)-β-D-glucopyranoside.
2. Results and discussion
Compound 3, obtained as a colorless solid, gave the molecular
formula of C20H34O12 determined by HRSIMS at m/z 489.1950 [M
+Na]+ (calcd for C20H34O12Na, 489.1948). The 1H and 13C NMR data
were similar to those of 2, but differed in the occurrence of char-
acteristic signals for a terminal double bond [δH 5.22 (1H, dd, J = 17.5,
1.5 Hz), 5.11 (1H, dd, J = 10.5, 1.5 Hz), 5.87 (1H, ddd, J = 17.5, 10.5,
8.0 Hz); δC 141.1, 117.0] in 3. Extensive analysis of 2D NMR data de-
The 95% and 50% aqueous EtOH extracts of the whole plant of O.
falcata was suspended in H2O and partitioned with petroleum ether,
CHCl3, EtOAc and n-BuOH, respectively. The n-BuOH soluble portion of
the 50% EtOH extract and the EtOAc soluble portion of the 95% and
50% EtOH extracts were subjected to repeated column chromatography
(CC) on Dianion HP20, MCI gel, Sephadex LH-20 and ODS-A, and
preparative thinner layer chromatography (PTLC) on silica gel to afford
five previously undescribed glycoconjugated volatile compounds (1–5)
and 11 known biological related ones (6–16) (Fig. 1).
duced
the
aglycone
of
3
to
be
CH2]CHeCH(O)
eCH2eCH2eCH2eCH2eCH3. Enzymatic hydrolysis yielded D-glucose
and D-glucuronic acid, and coupling patterns of anomeric protons
confirmed the β-configuration of the sugars. The glycosidic linkage as
determined by HMBC correlations were the same as in 2. 3R config-
uration was defined by optical rotation value of aglycone ([α]3D0 = −3.8
(c 0.16, CH2Cl2)). Consequently, compound 3 was characterized un-
ambiguously as (3R)-oct-1-ene-3-ol 3-O-β-D-glucuronic acid-(1 → 2)-β-
D-glucopyranoside.
Compound 1 was isolated as a colorless solid. Its molecular formula
was determined to be C20H36O12 by HRSIMS at m/z 469.2284 [M+H]+
(calcd for C20H37O12, 469.2285). The 1H and 13C NMR data exhibited
signals characteristic of an aliphatic chain [δH 0.91 (3H, t, J = 7.0 Hz),
1.33–1.39 (CH2 × 5), 1.65 (2H, quintet, J = 7.0 Hz); δC 13.4, 22.0,
25.1, 28.4, 28.5, 28.8, 31.1], an oxygenated methylene [δH 3.93 (1H,
m), 3.70–3.74 (1H, overlapped); δC 70.9], and two sugar units [δH 4.81
(1H, d, J = 8.0 Hz), 4.63 (1H, d, J = 8.0 Hz), 4.00–3.40 (multiple H,
overlapped); δC 102.2, 101.1, 80.7–60.7 (9 × C)]. The octan-1-ol unit
(eOCH2eCH2eCH2eCH2eCH2eCH2eCH2eCH3) was deduced from
the extensive analysis of 2D NMR data, especially those of 1H-1H COSY,
HSQC, and HMBC. Additionally, all the signals for sugar moiety were
totally assigned, and the characteristic carboxyl signal resonated at δC
175.5 was attributed to the sugar unit. The two sugars were identified
as Glc and GluA by enzymatic hydrolysis of 1 and then the following co-
TLC analysis, in which the Rf values of obtained sugar residues and
sugar standards were compared. Further purification of the sugar re-
sidues by CC on ODS-A yielded the purified Glc and GluA. D-config-
uration of the two sugars were determined according to the optical
rotation values ([α]D28 + 14.7 (c 0.19, H2O) for Glc and [α]2D8 + 10.9 (c
0.35, H2O) for GluA), and subsequently β-D-Glc and β-D-GluA were
confirmed according to the large coupling constants (J = 8.0 Hz) of
anomeric protons. The HMBC correlations of δH 4.63 (H-1′) and δC 70.9
(C-1), δH 4.81 (H-1″) and δC 80.7 (C-2′) indicated the C-1 linkage of
glucose to aglycone and C-2′ linkage of glucuronic acid to glucose. Thus
compound 1 was deduced to be octan-1-ol 1-O-β-D-glucuronic acid-
(1 → 2)-β-D-glucopyranoside.
Compound 2, isolated as a colorless solid, had the molecular for-
mula of C20H36O13 determined by HRSIMS at m/z 485.2233 [M+H]+
(calcd for C20H37O13, 485.2234). The 1H and 13C NMR features were
very similar with those of 1, except for the presence of one oxygenated
methine in 2 instead of one methylene in 1, indicating the presence of
one more hydroxyl group that was conformed to its molecular formula.
The aglycone was deduced to be octan-1, 3-diol (eOCH2eCH2eCH(O)
eCH2eCH2eCH2eCH2eCH3) by the extensive analysis of 2D NMR
data, and the sugar units were determined to be β-D-glucose and β-D-
glucuronic acid using the same method as described above. HMBC
correlations between δH 4.67 (H-1′) and δC 79.2 (C-3), and between δH
4.90 (H-1′′) and δC 79.7 (C-2′) defined the glycosidic linkage at C-3 and
Compound 4 was isolated as a colorless solid. Its molecular formula
was determined to be C22H36O12 by HRSIMS at m/z 515.2127 [M
+Na]+ (calcd for C22H36O12Na, 515.2104). Different from 1-3, com-
pound 4 was deduced to be a monoterpenoid disaccharide evidenced by
the characteristic 1H and 13C NMR signals for three methyls [δH 1.36
(3H, s), 1.60 (3H, s), 1.66 (3H, s); δC 17.9, 23.3, 25.9], two methylenes
[δH 1.63 (2H, m), 2.05 (2H, m); δC 41.1, 23.7], an oxygenated qua-
ternary carbon (δC 82.0), a terminal double bond [δH 5.18 (1H, dd,
J = 18.0, 1.0 Hz), 5.16 (1H, dd, J = 11.0, 1.0 Hz), 6.17 (1H, dd,
J = 18.0, 11.0 Hz); δC 115.1, 144.6], a trisubstituted double bond [δH
5.12 (1H, t-like, J = 7.0 Hz); δC 132.1, 125.9], and two sugar units [δH
4.58 (1H, d, J = 7.5 Hz), 4.48 (1H, d, J = 7.5 Hz), 3.80−3.10 (10H,
overlapped); δC 105.1, 98.0, 82.7−62.7 (10 × C)]. Further detailed
analysis of 2D NMR data confirmed the aglycone of 4 to be linalool. The
presence of β-D-glucose and β-D-glucuronic acid was defined based on
the enzymatic hydrolysis, comparison of the optical rotation value of
sugars and coupling patterns of anomeric protons. HMBC correlations
between δH 4.48 (H-1′) and δC 82.0 (C-3), and between δH 4.58 (H-1″)
and δC 82.7 (C-2′) determined the glycosylation of C-3 and C-2′. 3R
configuration was assigned by comparing the optical rotation value of
aglycone ([α]3D0 = −4.0 (c 0.10, CH2Cl2)) with that of R-(-)-linalool
(Konda et al., 1997). Thus the structure of compound 4 was identified
as (3R)-linalool 3-O-β-D-glucuronic acid-(1 → 2)-β-D-glucopyranoside.
Compound 5 was isolated as a colorless solid. Its molecular formula
was determined to be C22H32O12 by HRSIMS at m/z 489.1980 [M+H]+
(calcd for C22H33O12, 489.1972). The 1H and 13C NMR data of 5 were
similar to those of compounds 1-4 in the sugar units, but markedly
differed in the aglycone moiety. The presence of β-D-glucose and β-D-
glucuronic acid were confirmed by NMR data analysis and enzymatic
hydrolysis as described above. The aglycone of 5 was deduced to be 2-
hydroxy-4-phenyl-butane evidenced by the characteristic signals for a
monosubstituted phenyl [δH 7.12 − 7.23 (5H, overlapped); δC 144.0,
129.7 (2 × C), 129.4 (2 × C), 126.8], and 2,4-disubstituted butane [δH
144

S. Wang et al.
PhytochemistryLetters27(2018)143–147
1.24 (3H, d, J = 6.5 Hz), 1.76 − 2.70 (2 × CH2), 3.98 (1H, sextet,
J = 6.0 Hz); δC 20.0, 32.8, 40.3, 75.8], and confirmed by 2D NMR data.
2R configuration was assigned by comparing the optical rotation value
of aglycone ([α]3D0 = −5.5 (c 0.11, CH2Cl2)) with that reported in the
literature (Dai et al., 2005). Thus compound 5 was characterized as
(2R)-4-phenyl-butan-2-ol 2-O-β-D-glucuronic acid-(1 → 2)-β-D-gluco-
pyranoside.
The known compounds were identified as 3-hydroxyoctan-1-ol 3-O-
β-D-glucopyranoside (6) (Schwab et al., 1997), icariside C3 (7) (Miyase
et al., 1987), sammangaoside A (8) (Kanchanapooma et al., 2001),
sammangaoside B (9) (Kanchanapooma et al., 2001), benzyl-O-[β-D-
glucopyranosyl-(1 → 2)]-β-D-glucopyranoside (10) (Okamura et al.,
1981; Wu et al., 2004), 2-phenylethyl β-D-glucopyranoside (11)
(Yoneda et al., 2008), (3R)- -O-β-D-glucopyranosyloxy-5-phenylvaleric
acid (12) (Calis et al., 1999), zingerone 4-O-β-D-glucopyranoside (13)
(Ma et al., 2002), maltol 3-O-β-D-glucopyranoside (14) (Li and Huang,
2004), maltol 3-O-(6′-O-trans-p-coumaryl)-β-D-glucopyranoside (15) (Li
et al., 2005), and maltol 3-O-(6′-O-cis-p-coumaryl)-β-D-glucopyranoside
(16) (Yasue et al., 1970), by comparison of the spectral data with those
previously reported in the literatures. All the known compounds were
isolated from this herb and genus Oxytropis for the first time.
Chemical Co., Japan), MCI gel (20−45 μm, Fuji Silysia Chemical Co.,
Japan), ODS-A (50 μm, YMC Co. Ltd, Japan), and Sephadex LH-20 (GE
Healthcare Bio-Sciences AB, Uppsala, Sweden). Analytical and pre-
parative TLC was carried out on precoated silica gel 60 F254 plates
(Merck, Germany), and spots were visualized by 10% H2SO4/EtOH
reagent. All solvents used for extraction and isolation were of analytical
grade and purchased from Beijing Fine Chemicals (People’s Republic of
China) without further purification. All chemicals, unless otherwise
noted, were purchased from J & K Co. Ltd. (People’s Republic of China).
3.2. Plant material
The whole plant of O. falcata was collected from Guide County,
Qinghai Province, People’s Republic of China, and was authenticated by
Associate Professor Ying-Tao Zhang, Department of Natural Medicines,
School of Pharmaceutical Sciences, Peking University Health Science
Center. A voucher specimen (No. 20080801) was deposited in the
Herbarium of Department of Natural Medicines, School of
Pharmaceutical Sciences, Peking University Health Science Center.
3.3. Extraction and isolation
This is the first report of glycoconjugated volatile compounds with
structurally diverse aglycones, including octanols (1−3, 6), terpenoids
(4, 7−9), phenylbutanols (5, 12), phenylmethanol (10), phenylethanol
(11), phenylbutane-2-one (13) and chromonones (14−16), from O.
falcata. Wang et al had carried out investigation on the volatile com-
positions of O. falcata extracted by three different methods, including
water distillation, supercritical CO2 and headspace extraction techni-
ques, using GC and GC/MS analysis, and identified 58 compounds, in-
cluding paraffin hydrocarbons, flavonoids, aldehydes, terpenoids, or-
ganic acids, esters, and nitrogenous compounds. Of these, the main
components were paraffin hydrocarbons (30.1% for water distillation),
flavonoids (68.5% for supercritieal CO2 extraction) and aldehydes
(53.8% headspace extraction), indicative of much difference in the
volatile compositions extracted by different methods (Wang et al.,
2010). It is known that some volatile structures identified by GC–MS
may not be so reasonable and need more evidences to support. How-
ever, the volatile compounds are much difficult to obtain by traditional
chromatographic separation. So isolation and identification of glyco-
conjugated volatiles, which might be the precursor of free volatiles, can
provide more sound evidences and may be an alternative and feasible
means to elucidate free volatile compositions of herbs.
In conclusion, 16 glycoconjugated compounds with structurally di-
verse volatile aglycones but similar sugars were obtained from O. fal-
cata. Of these, five were previously undescribed compounds with dif-
ferent volatile aglycones but same glucuronic acid (1 → 2) glucosyl
moiety, and 11 were biological related known glucoconjugated volatile
compounds isolated from genus Oxytropis for the first time. The present
study is the first report of such structurally diverse glycoconjugated
volatile compounds from O. falcata, which might be regarded as the
precursor of free volatile compounds, and presents scientific evidences
for better clarifying the volatile compositions of this medicinal plant
and genus Oxytropis.
The dried whole plant of O. falcata (4.0 kg) was pulverized and in
turn percolated exhaustively with 95% EtOH and 50% EtOH. After
concentration, the 95% EtOH and 50% EtOH extracts were successively
partitioned with petroleum ether, CHCl3, EtOAc and n-BuOH, respec-
tively. The n-BuOH soluble part (136.0 g) of the 50% EtOH extract was
subjected to Dianion HP20 macroporous absorbent resin CC using an
EtOH/H2O gradient (0% → 95% EtOH) to yield six fractions (1–VI).
Fraction II (eluted with 20% EtOH, 4.0 g) was subjected to MCI gel CC
with a gradient system of MeOH/H2O (0% → 80% MeOH) to afford five
subfractions (DK2A–DK2E). Further purification of Dk2A (eluted with
H2O, 2.0 g) by repeated CC on Sephadex LH-20 (MeOH/H2O, 50%,
10%, and 0% MeOH), ODS-A (MeOH/H2O, 5% → 20%, 5% → 8% and
5%) and again on preparative TLC on silica gel (EtOAc-EtOH-H2O-
HCOOH, 8:3:1:1 and 10:2:1:1) yielded compounds 2 (5 mg), 3 (6 mg), 5
(14 mg), 12 (25 mg), and 14 (30 mg). Fraction DK2B (eluted with 20%
MeOH, 1.0 g) was subjected to repeated CC on Sephadex LH-20
(MeOH/H2O, 33% and 10% MeOH), ODS-A (MeOH/H2O, 5–25% and
23% MeOH) and again on preparative TLC on silica gel (EtOAc-EtOH-
H2O-HCOOH, 12:2:1:1) to give compounds 1 (5 mg), 4 (6 mg), and 10
(7 mg). Fraction DK2C (eluted with 40% MeOH, 1.0 g) was subjected to
repeated CC on Sephadex LH-20 (MeOH/H2O, 50%, 10%, and 5%),
ODS-A (MeOH/H2O, 10% → 35% MeOH) and again on preparative TLC
on silica gel (EtOAc-EtOH-H2O-HCOOH, 12:2:1:1) to afford compounds
8 (5 mg) and 9 (2 mg).
The EOAc soluble parts of the 95% and 50% EtOH extracts were
combined and then subjected to silica gel CC using an petrolum ether/
acetone gradient (10:1 → 1:5 and 95% EtOH) to yield six fractions
(E1–E6). Fraction E6 (eluted with petrolum ether/acetone 1:5 and 95%
EtOH, 25.0 g) was subjected to MCI gel CC with a gradient system of
MeOH/H2O (20% → 100% MeOH) to afford three subfractions (E6A-
E6C). Fraction E6B (eluted with 30% → 50% MeOH, 3.0 g) was sub-
jected to repeated CC on MCI gel (Acetone/H2O, 0–30% Acetone),
Sephadex LH-20 (MeOH/H2O, 10% → 40% MeOH), and ODS-A
(10% → 100% MeOH) to yield compounds 6 (4.1 mg), 7 (3.2 mg), 11
(2.2 mg), 12 (3.5 mg), and 13 (2.3 mg). Further purification of E6C
(eluted with (70% MeOH, 1.0 g) by repeated CC on Sephadex LH-20
(MeOH/H2O, 10% → 40% MeOH), and ODS-A (MeOH/H2O, 10% →
100% MeOH) afforded compounds 15 and 16 (3.5 mg).
3. Experimental section
3.1. General experimental procedures
Optical rotations were measured with
a Rudolph Research
Analytical Autopol III automatic polarimeter. IR spectra were recorded
on a Nicolet Nexus 470 FT-IR spectrophotometer using KBr pellets.
NMR spectra were recorded on a Varian INOVA-500 instrument.
Chemical shift values are given in δ (ppm) using the peak signals of
solvent as reference, and coupling constants are reported in Hz.
HRSIMS data were recorded on a Bruker APEX II FT-ICRMS spectro-
meter. CC was performed on Diaion HP20 (200−300 mesh, Mitsubishi
Compound 1. colorless solid; [α]3D0 − 26.4 (c 0.53, MeOH); IR (KBr)
νmax 3734, 3420, 2927, 2858, 1618, 1428, 1376, 1309, 1161, 1071,
951, 902, 723, 618, 460 cm−1 1H NMR (D2O, 500 MHz) and 13C NMR
;
(D2O, 125 MHz) data, see Table 1; HRSIMS (positive) m/z 469.2284 [M
+H]+ (calcd for C20H37O12, 469.2285).
Compound 2. colorless solid; [α]3D0 − 20.0 (c 0.70, MeOH); IR (KBr)
145

S. Wang et al.
PhytochemistryLetters27(2018)143–147
Table 1
1H (500 MHz) and 13C NMR (125 MHz) data of 1–3.
1a
2a
3b
position
1
δC
δH (J in Hz)
δC
δH (J in Hz)
δC
δH (J in Hz)
70.9
3.93, m
58.4
3.83, m, 3.79, m
117.0
5.22, dd (17.5, 1.5)
5.11, dd (10.5, 1.5)
5.87, ddd (17.5, 10.5, 8.0)
4.23, dd-like (13.5, 7.5)
1.69, m
3.70–3.74c
1.65, quintet (7.0)
1.33–1.39c
1.33–1.39c
2
3
4
28.8
25.1
28.5
35.7
79.2
34.2
1.85, m
3.96, m
1.70, m, 1.61, m
141.1
83.8
35.9
1.50, m
5
6
7
8
1'
2'
3'
4'
5'
6'
28.4
31.1
22.0
13.4
101.1
80.7
76.3
69.5
75.6
60.7
1.33–1.39c
24.0
31.0
21.9
13.3
100.6
79.7
76.4
69.6
75.5
60.7
1.33–1.42c
25.9
33.2
23.8
14.6
101.5
82.7
77.8
71.7
77.6
62.8
1.29–1.32c
1.33–1.39c
1.33–1.42c
1.29–1.32c
1.33–1.39c
1.33–1.42c
1.29–1.32c
0.91, t (7.0)
4.63, d (8.0)
3.58, t-like (9.5)
3.70– 3.74c
0.92, t (7.0)
4.67, d (8.0)
3.63, dd (9.0, 8.0)
3.73–3.77c
0.89, t (6.5)
4.52, d (7.5)
3.49, t-like (9.0)
3.55, t-like (9.0)
3.34, t-like (9.5)
3.19, ddd (9.5, 5.5, 3.0)
3.79, dd (12.0, 3.0)
3.64, dd (12.0, 5.5)
4.62, d (7.5)
3.45–3.50c
3.48–3.49c
3.45–3.50c
3.48–3.49c
3.96, dd (12.5, 1.5)
3.76, dd (12.5, 5.0)
4.81, d (8.0)
3.40, t-like (8.5)
3.55–3.57c
3.94, d-like (12.5)
3.73–3.77c
1”
2”
3”
4”
5”
6”
102.2
73.6
75.4
71.8
75.7
175.5
102.2
73.9
75.4
71.8
76.4
175.6
4.90, d (8.0)
3.39, td (8.0, 1.5)
3.56–3.58c
104.3
75.7
77.6
73.8
76.6
176.5
3.30-3.36c
3.43, t-like (7.0)
3.41, t-like (7.0)
3.58, d (8.0)
3.55–3.57c
3.56–3.58c
3.70–3.74c
3.73–3.77c
a
Recorded in D2O.
Recorded in CD3OD.
Signals overlapped.
b
c
νmax 3733, 3420, 2929, 2871, 1726, 1632, 1590, 1511, 1412, 1371,
1287, 1260, 1232, 1170, 1070, 1039, 943, 619, 552, 454 cm−1 1H
Table 2
1H (500 MHz) and 13C NMR (125 MHz) data of 4 and 5 in CD3OD.
;
NMR (D2O, 500 MHz) and 13C NMR (D2O, 125 MHz) see Table 1;
4
5
HRSIMS (positive) m/z 485.2233 [M+H]+ (calcd for C20H37O13
,
position δC
δH (J in Hz)
position δC
δH (J in Hz)
485.2234).
Compound 3. colorless solid; [α]2D8 − 23.6 (c 0.39, MeOH); IR (KBr)
νmax 3417, 2932, 1624, 1466, 1432, 1381, 1322, 1243, 1161, 1125,
1
115.1 5.18, dd (18.0, 1.0)
5.16, dd (11.0, 1.0)
144.6 6.17, dd (18.0, 11.0)
82.0
1
20.0
1.24, d (6.5)
1044, 891, 773, 662, 605, 462, 424 cm−1 1H NMR (CD3OD, 500 MHz)
;
2
3
2
3
75.8
40.3
3.98, sextet (6.0)
1.92, m
1.76, m
and 13C NMR (CD3OD, 125 MHz) data see Table 1; HRSIMS (positive)
m/z 489.1950 [M+Na]+ (calcd for C20H34O12Na, 489.1948).
Compound 4. colorless solid; [α]2D8 − 10.6 (c 0.53, MeOH); IR (KBr)
νmax 3734, 3421, 2927, 2833, 2721, 1604, 1415, 1363, 1168, 1071,
4
5
41.1
23.7
1.63, m
2.05, m
4
32.8
2.70, m
1'
2'
3'
4'
5'
6'
1”
2”
3”
4”
5”
144.0
6
7
125.9 5.12, t-like (7.0)
132.1
129.4 7.22–7.23a
129.7 7.22–7.23a
126.8 7.12, m
940, 876, 775, 614 cm−1 1H NMR (CD3OD, 500 MHz) and 13C NMR
;
(CD3OD, 125 MHz) data see Table 2; HRSIMS (positive) m/z 515.2127
8
9
17.9
25.9
23.3
98.0
82.7
77.8
71.7
77.4
1.60, s
1.66, s
1.36, s
4.48, d (7.5)
3.46, t-like (8.0)
3.52, t-like (9.0)
3.343, t-like (9.0)
3.14, ddd (9.5, 5.5,
2.5)
129.7 7.22–7.23a
129.4 7.22–7.23a
100.9 4.56, d (7.5)
82.4
77. 7
71.8
77.7
[M+Na]+ (calcd for C22H36O12Na, 515.2104).
Compound 5. colorless solid; [α]2D8 − 23.6 (c 0.25, MeOH); IR (KBr)
νmax 3443, 2926, 1631, 1494, 1455, 1414, 1362, 1165, 1112, 1064,
10
1'
2'
3'
4'
5'
3.48, dd (9.0, 7.5)
943, 902, 740, 698, 562 cm−1 1H NMR (CD3OD, 500 MHz) and 13C
;
3.58, t-like (9.0)
3.38, t-like (9.0)
3.27, ddd (10.0, 5.5,
2.0)
NMR (CD3OD, 125 MHz) data see Table 2; HRSIMS (positive) m/z
489.1980 [M+H]+ (calcd for C22H33O12, 489.1972),
6'
62.7
3.78, dd (12.0, 2.5)
3.65, dd (12.0, 5.5)
6”
62.8
3.86, dd (11.5, 2.0)
3.70, dd (11.5, 5.5)
3.4. Enzymatic hydrolysis and absolute configuration determination
1”
2”
3”
4”
5”
6”
105.1 4.58, d (7.5)
1”'
2”'
3”'
4”'
5”'
6”'
104.1 4.63, d (7.5)
75.7
77.4
73.6
77.0
176.5
3.336, t-like (8.0)
75.3
77.6
73.8
77.1
176.6
3.34, td (7.5, 2.0)
Compounds 1 (1.0 mg), 2 (4.4 mg), 3 (5.8 mg), 4 (3.2 mg), and 5
(3.7 mg) were mixed with 0.2 mL KH2PO4/KOH buffer (pH = 4.99) and
0.1 mL glucuronic acid hydrolyase solution (Type HP-2 from Helix
pomatia; Sigma-Aldrich), respectively. Then each solution was virtex
mixed, and maintained for 5 h at 37 °C. After stopping, each reaction
mixture was first extracted with CH2Cl2 to obtain the aglycone for
further analysis, and then MeOH–CH3CN (2:1) was added to precipitate
the protein. After centrifugation for 5 min at 13,500 rpm, the upper
solution was analyzed using TLC over silica gel (EtOAc-EtOH-H2O-
HCOOH, 6:4:1:1) by comparing with authentic sugars, proving the
presence of glucose (Glc) and glucuronic acid (GluA) in compounds
1−5. Then the sugar residues was combined and subjected to CC on
ODS-A eluted with H2O to yield Glc and GluA. The optical rotation
values of [α]2D8 + 14.7 (c 0.19, H2O) for Glc and [α]2D8 + 10.9 (c 0.35,
3.43–3.45a
3.43–3.45a
3.61, d (9.0)
3.44, t-like (7.0)
3.42, t-like (7.0)
3.60, d (7.5)
a
Signals overlapped.
H2O) for GluA confirmed the D-configurations of Glc and GluA.
The CH2Cl2 extract was evaporated to yield the aglycones of 2−5,
respectively, which were obtained as oils and smelled aromatic at room
temperature. The 3R configurations for 2−4 and 2R configuration for 5
were defined on the basis of the rotation value of [α]3D0 + 6.7 (c 0.27,
CH2Cl2) for the aglycone of 2, [α]3D0 − 3.8 (c 0.16, CH2Cl2) for the
aglycone of 3, [α]3D0 − 4.0 (c 0.10, CH2Cl2) for the aglycone of 4, and
146

S. Wang et al.
PhytochemistryLetters27(2018)143–147
[α]3D0 − 5.5 (c 0.11, CH2Cl2) for the aglycone of 5.
Conflict of interest statement
The authors have declared no conflict of interest.
Acknowledgements
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This project was financially supported by National Natural Science
Foundation of China (200872006, 21172008, and 21372015) and
National Key Technology R & D Program “New Drug Innovation” of
China (No. 2017ZX 09101003-008-003). We also sincerely thank Drs.
Qin Li and Xu-Lin Sun at Peking University Health Science Center for
measuring the NMR data.
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