Hydroquinone and terpene glucosides from Leontopodium leontopodioides and their lipase inhibitory activity

Article abstract of DOI:10.1016/j.fitote.2018.08.010

Three new glucosides of hydroquinone, monoterpene, and megastigmane, benzyl 2,5-dihydroxybenzoate 5-O-β-D-glucopyranoside (isotrichocarpin, 1), (2S,3R)-3,7-dimethyl-6-octene-1,2,3-triol 2-O-β-D-glucopyranoside (leontopodioside D, 4), and (6R,7R,8R,9S)-6,9-epoxy-7,8-dihydroxymegastigman-4-en-3-one 8-O-β-D-glucopyranoside (leontopodioside E, 7) were isolated from the whole herbs of Leontopodium leontopodioides (Willd.) Beauv. (Asteraceae), along with nebrodenside A (2), pungenin (3), betulalbuside A (5), geranyl O-β-D-glucopyranoside (6), and 3β-hydroxy-β-ionone 3-O-β-D-glucopyranoside (8). Their structure were determined by spectroscopic and chemical methods. All the known compounds were reported from this species for the first time. Compounds 2–6 showed potent in vitro pancreatic lipase inhibitory activity, suggesting their participation in the reductive effect of the herbs on triglyceride absorption.

Full text of DOI:10.1016/j.fitote.2018.08.010

Fitoterapia130(2018)89–93
Contents lists available at ScienceDirect
Fitoterapia
journal homepage: www.elsevier.com/locate/fitote
Hydroquinone and terpene glucosides from Leontopodium leontopodioides
and their lipase inhibitory activity
Ping Goua,⁎, Yangyang Xiaob,c, Ling Lvb,c, Haihui Xieb,c,
⁎⁎
a
College of Life Science and Technology, Xinjiang University, Urumqi 830046, China
Key Laboratory of South China Agricultural Plant Molecular Analysis and Genetic Improvement, Guangdong Provincial Key Laboratory of Applied Botany, South China
b
Botanical Garden, Chinese Academy of Sciences, Guangzhou, China
c
University of Chinese Academy of Sciences, Beijing 100049, China
A R T I C L E I N F O
A B S T R A C T
Keywords:
Three new glucosides of hydroquinone, monoterpene, and megastigmane, benzyl 2,5-dihydroxybenzoate 5-O-β-
D-glucopyranoside (isotrichocarpin, 1), (2S,3R)-3,7-dimethyl-6-octene-1,2,3-triol 2-O-β-D-glucopyranoside
(leontopodioside D, 4), and (6R,7R,8R,9S)-6,9-epoxy-7,8-dihydroxymegastigman-4-en-3-one 8-O-β-D-glucopyr-
anoside (leontopodioside E, 7) were isolated from the whole herbs of Leontopodium leontopodioides (Willd.)
Beauv. (Asteraceae), along with nebrodenside A (2), pungenin (3), betulalbuside A (5), geranyl O-β-D-gluco-
pyranoside (6), and 3β-hydroxy-β-ionone 3-O-β-D-glucopyranoside (8). Their structure were determined by
spectroscopic and chemical methods. All the known compounds were reported from this species for the first
time. Compounds 2–6 showed potent in vitro pancreatic lipase inhibitory activity, suggesting their participation
in the reductive effect of the herbs on triglyceride absorption.
Leontopodium leontopodioides
Hydroquinone glucoside
Monoterpene glucoside
Megastigmane glucoside
Pancreatic lipase inhibition
1. Introduction
to a 2-monoglyceride and two fatty acids. They are the major enzymes
involved in the hydrolysis of carbohydrates and fat. Therefore, α-glu-
Leontopodium leontopodioides (Willd.) Beauv. (Asteraceae) is per-
ennial herbs growing at the altitudes of 100–3800 m in northwest and
northeast regions of China, Mongolia, Russia, Korea, and Japan [1]. The
herbs have been used in Chinese medicinal formulae to treat influenza,
acute or chronic nephritis, urinary tract infection, hematuria, and
trauma hemorrhage [2]. Previous pharmacological studies revealed
that the herbs possess anti-inflammatory, antibacterial, and hepato-
protective activities. Chemical studies clarified diverse structural types
of compounds, including flavone, flavonol, flavanonol, phenylpropa-
noid, isobenzofuranone, and ent-kaurene diterpenoid [2–6]. Liu et al.
(2014) clarified that the decoction of the herbs could significantly re-
duce the levels of blood glucose, triglyceride, and cholesterol, and in-
crease serum insulin level in type 2 diabetic rats [7]. Our pharmaco-
logical study showed that the ethanol extract of the whole herbs could
remarkably reduce the blood glucose level of hyperglycemia mice [8].
That inspired us to investigate the chemical compounds present in the
herbs with hypoglycemic or lipid disorder improving activities. Alpha-
glucosidase breaks down starch and disaccharides to glucose and other
monosaccharides, and pancreatic lipase rapidly converts a triglyceride
cosidase and pancreatic lipase become a conventional approach for the
search of natural inhibitors of carbohydrates and triglyceride absorp-
tion [9]. As a result, three new acyl flavone and lignan glucosides with
potent α-glucosidase inhibitory activity were previously obtained from
the whole herbs [10]. Further study led to the isolation of eight glu-
cosides of hydroquinone (1 and 2), acetophenone (3), monoterpene
(4–6), and megastigmane (7 and 8), of which compounds 1, 4, and 7
were new structures and the others were obtained from this species for
the first time (Fig. 1).
2. Experimental
2.1. General procedures
Silica gel (100–200 mesh, Qingdao Haiyang Chemical Co.,
Shandong, China) and Sephadex LH-20 (GE Healthcare Bio-Sciences
AB, Uppsala, Sweden) were used for column chromatography (CC).
Thin layer chromatography (TLC) was conducted on pre-coated silica
gel HSGF254 plates (Jiangyou Silica Gel Development Co., Yantai,
⁎
Corresponding author.
⁎⁎
Corresponding author at: Key Laboratory of South China Agricultural Plant Molecular Analysis and Genetic Improvement, Guangdong Provincial Key Laboratory
of Applied Botany, South China Botanical Garden, Chinese Academy of Sciences, Guangzhou, China.
E-mail addresses: gou_ping@sina.com (P. Gou), xiehaih@scbg.ac.cn (H. Xie).
https://doi.org/10.1016/j.fitote.2018.08.010
Received 15 July 2018; Received in revised form 18 August 2018; Accepted 22 August 2018
Availableonline23August2018
0367-326X/©2018PublishedbyElsevierB.V.

P. Gou et al.
Fitoterapia130(2018)89–93
Fig. 1. Structures of compounds 1–8, 4a, and 7a.
China) and visualized under UV light (λ = 254 nm) and then by heating
after sprayed 10% sulfuric acid in ethanol (v/v). Medium pressure li-
quid chromatography (MPLC) was performed on an EZ Purifier (Lisure
Science, Suzhou, China) and the column used was 400 mm × 25 mm
i.d., Chromatorex RP-18 SMB100, particle size 20–45 μm (Shanghai
Lisui E-Tech Co., Shanghai, China). HPLC was run on a LC-6AD liquid
chromatograph (Shimadzu, Kyoto, Japan) connected to a RID-10A re-
fractive index detector (Shimadzu) and the columns used were
250 mm × 4.6 mm i.d., Cosmosil 5C18-MS-II, 5 μm (Nacalai Tesque,
Inc., Kyoto, Japan) for analysis and 250 mm × 20 mm i.d., YMC-Pack
ODS-A, 5 μm (YMC Co., Kyoto, Japan) for preparation. 1H and 13C NMR
spectra were measured on a Bruker Ascend-500 spectrometer in CD3OD
or acetone-d6 (Sigma-Aldrich, St. Louis, MO, USA) using solvent re-
sidual peaks as references or in CDCl3 using tetramethylsilane (TMS) as
reference. 1He1H COSY, HSQC, HMBC, and NOESY spectra were
measured on the same spectrometer as necessary. HR-ESI-MS and ESI-
MS spectra were performed on Bruker maXis mass spectrometer and
MDS SCIEX API 2000 LC/MS/MS apparatus (Applied Biosystems Inc.,
Forster, CA, USA). Optical rotations (αD) and UV spectra were acquired
on a Perkin-Elmer 343 polarimeter and a Lambda 650 UV/Vis spec-
trometer (Perkin-Elmer, Waltham, MA, USA). IR spectra were measured
on an FT-IR Affinity-1 spectrometer (Shimadzu). CD spectra were re-
(115.87 g) and n-butanol soluble (198.68 g) fractions. The n-butanol
soluble fraction was separated by silica gel CC eluted with CHCl3/
MeOH (95:5–6:4, v/v) to furnish fractions 1–12 according to their TLC
profiles. Fraction 5 (2.24 g) was separated by MPLC eluted with MeOH/
H2O (1:9–10:0, v/v) to yield fractions 5–1–5-12. Fraction 5–6 was
purified by HPLC eluted with MeOH/H2O (33:67, v/v) at the flow rate
of 6 mL/min to provide compound 8 [retention time (tR) = 75 min,
7 mg]. Fraction 5–10 was purified by HPLC eluted with MeOH/H2O
(48:52, v/v) at 6 mL/min to yield compound 6 (tR = 88 min, 4 mg).
Fraction 6 (4.92 g) was separated by MPLC eluted with MeOH/H2O
(1:9–10:0, v/v) to afford fractions 6–1–6-13. Fraction 6–9 was purified
by HPLC eluted with MeOH/H2O (50:50, v/v) at 6 mL/min to furnish
compound 1 (tR = 76 min, 9 mg). Fraction 7 (11.03 g) was separated by
MPLC eluted with MeOH/H2O (1:9–7:3, v/v) to give fractions 7–1–7-
15. Fraction 7–4 was separated by Sephadex LH-20 CC eluted with
MeOH to provide compound 5 (6 mg). Fraction 7–5 was purified by
HPLC eluted with MeOH/H2O (30:70, v/v) at 6 mL/min to yield com-
pound 2 (tR = 95 min, 24 mg). Fraction 7–6 was purified by HPLC
eluted with MeOH/H2O (34:66, v/v) at 6 mL/min to furnish compound
7 (tR = 70 min, 3 mg). Fraction 7–7 was purified by HPLC eluted with
MeOH/H2O (34:66, v/v) at 6 mL/min to obtain compound
4
(tR = 88 min, 4 mg). Fraction 9 (9.63 g) was separated by MPLC eluted
with MeOH/H2O (1:9–10:0, v/v) to afford fractions 9–1–9-12. Fraction
9–3 was purified by HPLC eluted with MeOH/H2O (10:90, v/v) at
6 mL/min to give compound 3 (tR = 58 min, 10 mg).
corded on
a Chirascan circular dichroism spectrometer (Applied
Photophysics Ltd., Surrey, UK).
2.2. Plant material
2.4. Spectroscopic data of compounds 1, 4, 7, and 8
Fresh whole herbs of Leontopodium leontopodioides (Willd.) Beauv.
(Asteraceae)
were
collected
from
Miaoergou
village
2.4.1. Isotrichocarpin (1)
(87°44′20"–87°44′41" E and 43°44′13"–43°44′16" N) at the altitudes of
1760–1800 m, Shuixi Town, Urumqi County, Xinjiang Uygur
Autonomous Region in late June of 2013 and botanically authenticated
by associate professor F.Y. Suo and laboratory technician Y.M. Lu,
College of Life Science and Technology, Xinjiang University. A voucher
specimen (No. 13062) was deposited at the Herbarium of Xinjiang
University.
White amorphous powder (MeOH); [α]D20–25.6 (c 0.22, MeOH); UV
(MeOH) λmax nm (log ε) 211 (4.27), 237 (3.70), and 323 (3.40); IR
(MeOH) νmax 3377, 2924, 1684, 1489, 1456, 1213, 1072, 1018, 730,
and 692 cm−1; ESI-MS m/z 429.4 [M + Na]+, 404.8 [M – H]−, and
441.1 [M + Cl]−; HR-ESI-MS m/z 441.0969 [M + Cl]− (calcd for
C
20H22O9Cl− 441.0958, error − 1.1 mDa); 1H (500 MHz) and 13C
(125 MHz) NMR data in CD3OD and acetone-d6, see Table 1.
2.3. Extraction and isolation
2.4.2. Leontopodioside D (4)
Air-dried herbs were ground to powder. The powder (7270 g) was
immersed in 95% ethanol (60 L and 50 L) twice and 50% ethanol (50 L)
once for 2 d per time. The combined filtrate was condensed under va-
cuum to yield an ethanol extract (1414.50 g). The extract was dissolved
in 3 L of distilled water and fractionated with CHCl3 for four times
(3 L × 4) and n-butanol for five times (3 L × 5) to give CHCl3 soluble
White amorphous powder (MeOH); [α]D20–19.0 (c 0.30, MeOH); UV
(MeOH) λmax nm (log ε) 210 (3.47); IR (MeOH) νmax 3277, 2924, 1645,
1072, and 1028 cm−1; ESI-MS m/z 373.2 [M + Na]+, 349.3 [M – H]−
,
and 385.3 [M + Cl]−; HR-ESI-MS m/z 373.1837 [M + Na]+ (calcd for
16H30O8Na+ 373.1833, error − 0.4 mDa); 1H (500 MHz) and 13C
(125 MHz) NMR data in CD3OD, see Table 2.
C
90

P. Gou et al.
Fitoterapia130(2018)89–93
Table 1
d, J = 16.4 Hz, H-8), 4.44 (1H, d, J = 7.8 Hz, H-1′), 4.10 (1H, m, H-3),
3.87 (1H, dd, J = 11.9, 2.0 Hz, H-6′), 3.68 (1H, dd, J = 11.9, 5.1 Hz, H-
6′), 3.16 (1H, dd, J = 9.2, 7.8 Hz, H-2′), 2.56 (1H, dd, J = 17.8, 5.6 Hz,
H-4), 2.30 (3H, s, H3–10), 2.17 (1H, dd, J = 17.8, 5.6 Hz, H-4), 1.81
(3H, s, H3–13), 1.15 (3H, s, H3–12), and 1.12 (3H, s, H3–11); 13C NMR
(CD3OD, 125 MHz) δ 37.7 (C-1), 47.3 (C-2), 72.5 (C-3), 40.4 (C-4),
133.2 (C-5), 137.1 (C-6), 144.4 (C-7), 134.0 (C-8), 201.2 (C-9), 27.2 (C-
10), 30.5 (C-11), 28.7 (C-12), 21.8 (C-13), 102.5 (C-1′), 75.2 (C-2′),
78.1 (C-3′), 71.7 (C-4′), 77.9 (C-5′), and 62.7 (C-6′). This was the first
report of its NMR data measured in CD3OD.
1H and 13C NMR data of compound 1.
a
a
b
b
No.
δH (mult., J in Hz)
δC
δH (mult., J in Hz)
δC
1
2
113.4, C
158.4, C
117.9
157.9
118.9
126.9
151.2
118.9
170.4
136.6
129.2
129.5
129.3
129.5
129.2
67.9
3
6.89 (d, 9.1)
119.1, CH
127.3, CH
151.5, C
118.4, CH
170.7, C
6.91 (d, 9.0)
7.34 (dd, 9.0, 3.0)
4
7.32 (dd, 9.1, 3.1)
5
6
7.62 (d, 3.1)
7.58 (d, 3.0)
7
1′
2′
3′
4′
5′
6′
7′
137.0, C
7.48 (dt, 7.4, 1.8)
7.40 (tt, 7.4, 1.8)
7.36 (tt, 7.4, 1.8)
7.40 (tt, 7.4, 1.8)
7.48 (dt, 7.4, 1.8)
5.41 (d, 12.3)
5.37 (d, 12.3)
4.75 (d, 7.0)
3.42 (m)
3.42 (m)
3.41 (m)
3.35 (m)
3.80 (dd, 12.1, 2.3)
3.69 (dd, 12.1, 5.0)
129.4, CH
129.7, CH
129.5, CH
129.7, CH
129.4, CH
68.2, CH2
7.55 (br d, 7.1)
7.44 (br t, 7.1)
7.38 (br t, 7.1)
7.44 (br t, 7.1)
7.55 (br t, 7.1)
5.46 (d, 12.6)
5.42 (d, 12.6)
4.83 (d, 7.5)
3.46 (m)
3.46 (m)
3.46 (m)
3.42 (m)
3.84 (br d, 11.6)
3.70 (br d, 11.6)
2.5. β-Glucosidase hydrolysis of compounds 4 and 7
β-Glucosidase hydrolysis was conducted following our previous
procedures [11]. Briefly, the aqueous solution of compound 4 (2.8 mg
in 1.0 mL of distilled water) was added 2 mg of β-glucosidase (from
Almond, Sigma-Aldrich Co., St. Louis, MO, USA), stirred at 37 °C for 4 d,
and then fractionated with EtOAc thrice (1 mL per time). After dehy-
drated with anhydrous sodium sulfate and filtrated, the combined
EtOAc solution was concentrated under vacuum to give 4a (1.4 mg,
93% yield). Through the same procedures, compound 7a (1.1 mg, 92%
yield) was obtained from 7 (2.0 mg).
1″
2″
3″
4″
5″
6″
103.7, CH
74.9, CH
77.9, CH
71.1, CH
78.1, CH
62.3, CH2
103.2
74.6
77.8
71.2
77.8
62.5
a
In CD3OD.
In acetone-d6.
2.5.1. (2S,3R)-3,7-Dimethyl-6-octene-1,2,3-triol (4a)
b
White amorphous powder (CHCl3); [α]D20–12.5 (c 0.12, CHCl3);
ESI-MS m/z 211.1 [M + Na]+, 227.1 [M + K]+ 1H NMR (CDCl3,
;
Table 2
1H and 13C NMR data of compounds 4 and 7 in CD3OD.
500 MHz) δ 5.12 (1H, tt, J = 6.8, 1.4 Hz, H-6), 3.78 (2H, d, J = 4.8 Hz,
H2–1), 3.49 (1H, t, J = 4.8 Hz, H-2), 2.14, 2.05 (each 1H, tt, J = 12.4,
6.8 Hz, H2–5), 1.69 (3H, s, H3–8),1.64 (1H, m, H-4), 1.63 (3H, s, H3–9),
1.43 (1H, ddd, J = 14.0, 10.9, 5.6 Hz, H-4), and 1.25 (3H, s, H3–10);
13C NMR (CDCl3, 125 MHz) δ 63.2 (C-1), 76.2 (C-2), 74.7 (C-3), 37.9
(C-4), 22.2 (C-5), 124.0 (C-6), 132.3 (C-7), 25.7 (C-8), 17.7 (C-9), and
23.5 (C-10).
4
7
No.
1
δH (mult., J in Hz)
δC
δH (mult., J in Hz)
δC
3.77 (dd, 12.0, 2.6)
3.63 (dd, 12.0, 7.4)
3.52 (dd, 7.4, 2.6)
63.4, CH2
90.9, CH
41.6, C
2
1.97 (d, 17.9, 1.3)
3.40 (d, 17.9)
49.7, CH2
2.5.2. (6R,7R,8R,9S)-6,9-Epoxy-7,8-dihydromegastigman-4-en-3-one
(7a)
3
4
75.0, C
38.8, CH2
202.3, C
126.5, CH
1.63 (m)
1.45 (ddd,13.9, 11.7, 5.4)
2.12 (m)
2.05 (m)
5.12 (tt, 7.2, 1.5)
5.83 (br t, 1.3)
White amorphous powder (CHCl3); [α]D20 + 12.8 (c 0.11, MeOH);
UV (MeOH) λmax nm (log ε) 231 (3.82); ESI-MS m/z 241.3 [M + H]+
,
5
23.0, CH2
169.4, C
263.3 [M + Na]+, 279.0 [M + K]+, and 275.1 [M + Cl]−; CD (MeOH)
Δε 231 (−0.90) and 263 (+1.52); 1H NMR (CDCl3, 500 MHz) δ 5.83
(1H, t, J = 1.4 Hz, H-4), 4.21 (H, d, J = 8.7 Hz, H-7), 4.00 (1H, t,
J = 8.7 Hz, H-8), 3.80 (1H, dq, J = 8.7, 6.1 Hz, H-9), 3.24 (1H, d,
J = 18.1 Hz, H-2), 2.06 (1H, d, J = 18.1 Hz, H-2), 2.02 (3H, s, H3–13),
1.37 (1H, d, J = 6.0 Hz, H3–10), 1.19 (3H, s, H3–11), 0.99 (3H, s,
H3–12); 13C NMR (CDCl3, 125 MHz) δ 40.4 (C-1), 48.7 (C-2), 199.9 (C-
3), 126.0 (C-4), 166.4 (C-5), 85.0 (C-6), 84.9 (C-7), 80.1 (C-8), 75.6 (C-
9), 18.2 (C-10), 25.8 (C-11), 25.5 (C-12), and 18.5 (C-13).
6
7
8
9
125.8, CH
132.1, C
25.9, CH3
17.7, CH3
22.9, CH3
86.3, C
4.36 (d, 8.5)
4.08 (t, 8.5)
3.91 (dq, 8.5, 6.0)
1.40 (3H, d, 6.0)
1.24 (3H, s)
0.97 (3H, s)
2.04 (3H, d, 1.3)
4.58 (d, 7.8)
3.19 (dd, 7.8, 8.8)
3.35 (t, 8.8)
3.33 (overlap)
3.28 (m)
85.0, CH
88.5, CH
76.3, CH
18.7, CH3
25.9, CH3
26.1, CH3
18.7, CH3
103.7, CH
75.2, CH
78.0, CH
71.4, CH
77.9, CH
62.6, CH2
1.67 (3H, s)
1.62 (3H, s)
1.19 (3H, s)
10
11
12
13
1′
2′
3′
4′
5′
6′
4.38 (d, 7.8)
3.27 (dd, 7.8, 9.0)
3.37 (t, 9.0)
3.29 (m)
3.32 (m)
106.0, CH
75.5, CH
78.1, CH
71.6, CH
78.0, CH
62.6, CH2
2.6. Determination of glucose absolute configuration
3.88 (dd, 11.8, 2.2)
3.64 (dd, 11.8, 5.6)
3.87 (dd, 12.0, 2.2)
3.70 (dd, 12.0, 5.1)
Glucose absolute configuration was determined following our pre-
vious procedures [10]. In brief, compound 1 (1 mg) was dissolved in
5 mL of 2 M aqueous HCl and refluxed at 90 °C for 4 h. After removal of
the solution under vacuum, the residue was dissolved in 5 mL of water,
and partitioned with 5 mL of EtOAc thrice. The aqueous layers of
compound 1 from acid hydrolysis and of compounds 4 and 7 from β-
glucosidase hydrolysis were condensed, and each residue was dissolved
in 1 mL of pyridine containing 1 mg/mL L-cystein methyl ester hydro-
chloride (Shanghai Macklin Biochemical Co., China). After the solution
was heated at 60 °C for 1 h, 2 μL of O-tolylisothiocyanate (Tokyo Che-
mical Industry Co., Japan) was added and kept at 60 °C for 1 h, and then
condensed under vacuum. Each residue was dissolved in 1 mL of MeOH
and analyzed by HPLC at 40 °C on a Prominence LC-20AT connected to
a SPD-M20A diode array detector and a CTO-20A column oven (Shi-
madzu) at 254 nm, and the column used was Cosmosil 5C18-MS-II using
CH3CN/H2O/acetic acid (22:78:0.1, v/v/v) as mobile phase at the flow
2.4.3. Leontopodioside E (7)
White amorphous powder (MeOH); [α]D20–7.3 (c 0.30, MeOH); UV
(MeOH) λmax nm (log ε) 242 (3.99); IR (MeOH) νmax 3288, 2924, 1771,
1645, 1033, and 665 cm−1; CD (MeOH) Δε 221 (−5.11) and 266
(+0.31); ESI-MS m/z 425.4 [M + Na]+, 401.4 [M – H]−, and 439.5
[M + Cl]−
C
;
HR-ESI-MS m/z 425.1768 [M + Na]+ (calcd for
19H30O9Na+ 425.1782, error + 1.4 mDa); 1H (500 MHz) and 13C
NMR (125 MHz) data in CD3OD, see Table 2.
2.4.4. 3β-Hydroxy-β-ionone 3-O-β-D-glucopyranoside (8)
White amorphous powder (MeOH); [α]D20–22.4 (c 0.29, MeOH);
ESI-MS m/z 393.6 [M + Na]+, 409.6 [M + K]+, and 405.1 [M + Cl]−
;
1H NMR (CD3OD, 500 MHz) δ 7.33 (1H, d, J = 16.4 Hz, H-7), 6.14 (1H,
91

P. Gou et al.
Fitoterapia130(2018)89–93
Fig. 2. Key 2D-NMR correlations of compounds 1, 4, and 7.
rate of 0.8 mL/min for 60 min, and then washed with 90% aqueous
CH3CN. Authentic D-glucose and L-glucose (Aladdin Industrial Corp.,
Shanghai, China) were treated in the same procedures.
The molecular formula C19H30O9 of compound 7 was deduced from
its HR-ESI-MS and NMR data. Except for the signals of six carbons
readily recognized for a β-glucosyl moiety in the 1H and 13C NMR
spectra (Table 2), the remaining thirteen carbons including four methyl
groups (CH3–10–13) were typical of a megastigmane skeleton [18]. In
combination with the HSQC spectrum, the presence of a carbonyl (C-3),
an olefinic bond (CH-4 and C-5), an oxygenated quaternary carbon (C-
6), and three oxygenated methines (C-7–9) were established. Analyses
of the HMBC and COSY spectra (Fig. 2) elucidated its planar structure of
6,9-epoxy-7,8-dihydromegastigman-4-en-3-one, which was previously
reported from Kalanchoe tubiflora (Crassulaceae) [19] and Sonneratia
ovata (Sonneratiaceae) [18]. The HMBC correlations from the anomeric
proton (H-1′) to C-8 and H-8 to C-1′ ascertained the connection of β-
glucosyl moiety to C-8. Moreover, The NOESY correlations between H-7
and H-9, H-7 and H3–11, H-7 and H3–13, H-8 and H3–10, H-9 and
H3–13 indicated that H-7 and H-9 were in β-orientation and H-8 was in
α-orientation. That is to say, the relative configurations of H-7, H-8, and
H-9 of 7 are the same as tubiflorone, which was also confirmed by the
consistence of their J values [19]. Further, β-glucosidase hydrolysis of 7
afforded its aglycone 7a, which showed a positive Cotton effect at
263 nm and so C-6 was assigned the R absolute configuration [20].
Hence, compound 7 was identified as (6R,7R,8R,9S)-6,9-epoxy-7,8-di-
hydromegastigman-4-en-3-one 8-O-β-D-glucopyranoside, and trivially
named leontopodioside E.
2.7. Assays for enzyme inhibitory activities
Assays for α-glucosidase and pancreatic lipase inhibitory activities
were conducted following our previous methods [10,12].
3. Results and discussion
Compound 1 was determined to have the molecular formula
C
20H22O9 based on its HRESIMS and NMR data. The 1H and 13C NMR
spectra (Table 1) exhibited signals of three ABX-coupled aromatic
protons (H-3, H-4, and H-6), five aromatic carbons of a mono-sub-
stituted phenyl (H-2′–6′), twelve aromatic carbons (C-1–6 and C-1′–6′),
a carboxy (C-7), an isolated oxygenated methylene (CH2–7′), and a β-
glucosyl moiety. With the aid of the HSQC spectrum, direct connections
of protons to carbons were assigned. The HMBC correlation (Fig. 2)
from H2–7′ to C-7 revealed the presence of a benzyl benzoate skeleton
[13]. However, comparison of its NMR data with those of trichocarpin
(both in acetone‑d6) found that there were obvious differences in H-1–6
and C-1–6 [14]. Further analyses of the HMBC spectrum clarified the
connection of glucosyl moiety to C-5 instead of to C-2 in trichocarpin.
Finally, acid hydrolysis of 1 liberated D-glucose, which was identified
by comparison of its tR value with those of authentic D-glucose
(tR ≈ 26 min) and L-glucose (tR ≈ 24 min) (Fig. S30). Therefore, com-
pound 1 was identified as benzyl 2,5-dihydroxybenzoate 5-O-β-D-glu-
copyranoside, i.e., isotrichocarpin.
The known compounds were identified as nebrodenside A (2) [21],
pungenin (3) [22], betulalbuside A (5) [23], geranyl O-β-D-glucopyr-
anoside (6) [24], and 3β-hydroxy-β-ionone 3-O-β-D-glucopyranoside
(8) [25] by analyses of their spectroscopic data and comparison of the
data with the reported values.
All the compounds except 7 (little quantity after enzymatic hydro-
lysis) were tested for in vitro inhibitory activities against α-glucosidase
from Saccharomyces cerevisiae Type I, lyophilized powder, ≥10 units/
mg protein (using p-nitrophenyl α-D-glucoside as substrate (Sigma-
Aldrich) and lipase from porcine pancreas Type II, 100–500 units/mg
protein (using olive oil (30 min incubation)), 30–90 units/mg protein
(using triacetin) (Sigma-Aldrich). As shown in Table 3, their inhibitory
rates against α-glucosidase were very low at the concentration of
100 μM, i.e., they did not exhibit α-glucosidase inhibitory activity.
Whereas compounds 2–6 demonstrated potent lipase inhibitory activity
Compound 4 was assigned the molecular formula C16H30O8 from its
m/z peak of [M + Na]+ in the HR-ESI-MS spectrum. The 1H and 13C
NMR and HSQC spectra (Table 2) showed signals of three methyl
groups (CH3–8–10), an olefinic bond (CH-6 and C-7), and a β-glucosyl
moiety, which suggested a monoterpene aglycone. The HMBC correla-
tions (Fig. 2) from H2–1 to C-3 and C-2, H-2 to C-4, C-10, and C-1,
H3–10 to C-2, C-4, and C-3, H3–8 and H3–9 to C-6 and C-7, H-6 to C-4,
in combination with the δ values of C-1, C-2, and C-3, led us to elucidate
the aglycone of 3,7-dimethyl-6-octene-1,2,3-triol [15]. In addition, the
HMBC correlations from H-2 to C-1′ and H-1′ to C-2 clarified the con-
nection of glucosyl moiety to C-2. Further, β-glucosidase hydrolysis of 4
yielded its aglycone 4a with negative optical rotation ([α]D20 = −12.5
in CHCl3), which indicated 2S,3S or 2S,3R absolute configurations
[15–17]. Comparison of the [α]D, 1H, and 13C NMR data of 4a with
those of (2S,3S)- and (2S,3R)-3,7-dimethyl-6-octene-1,2,3-triol led us to
determine the 2S,3R absolute configurations. Finally, from the aqueous
layer of β-glucosidase hydrolysis of 4, D-glucose was identified (Fig.
S30). Thus, compound 4 was identified as (2S,3R)-3,7-dimethyl-6-oc-
tene-1,2,3-triol 2-O-β-D-glucopyranoside, and trivially named leonto-
podioside D.
with the IC50 values of 12.3
0.9, 10.9
1.6, 41.2
4.7,
12.2 0.8, and 75.7 4.9 μM, respectively.
4. Conclusion
This study clarified the structures of three new and five known
glucosides of hydroquinone, acetophenone, monoterpene, and mega-
stigmane in the whole herbs of Leontopodium leontopodioides
(Asteraceae), and all the known compounds were previously un-
described from this species. Potent in vitro pancreatic lipase inhibitory
92

P. Gou et al.
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Table 3
Pancreatic lipase and α-glucosidase inhibitory activities of compounds 1–6 and
8.
Compound
IC50 (μM)
Lipase
Inhibitory rate (%) at 100 μM
α-Glucosidase
1
2
3
4
5
6
8
> 100
4.0
6.1
6.0
1.9
11.4
2.4
4.0
0.0
0.0
0.0
0.0
0.1
0.0
0.0
12.3
10.9
41.2
12.2
75.7
> 100
0.28
0.9
1.6
4.7
0.8
4.9
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Value represents mean
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Conflict of interest
The authors declare no conflict of interest.
Acknowledgements
The authors thank the Regional Science Foundation of National
Natural Science Foundation of China (No. 31460081) and Guangdong
Applied Science and Technology Research and Development Program
(2016B020239004) for financial support.
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