Lanostane Triterpenoids with PTP1B Inhibitory and Glucose-Uptake Stimulatory Activities from Mushroom Fomitopsis pinicola Collected in North America

Article abstract of DOI:10.1021/acs.jafc.0c04460

A chemical investigation on the fruiting bodies of Fomitopsis pinicola led to the isolation and identification of 28 lanostane triterpenoids including 11 new compounds (1-11) and 17 known analogues (12-28). Their structures were elucidated by extensive on

Full text of DOI:10.1021/acs.jafc.0c04460

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Bioactive Constituents, Metabolites, and Functions
Lanostane Triterpenoids with PTP1B Inhibitory and
Glucose-Uptake Stimulatory Activities from Mushroom
Fomitopsis pinicola Collected in North America
Jinjin Zhang, Baosong Chen, Jack Liang, Junjie Han,
Liwei Zhou, Ruilin Zhao, Hongwei Liu, and Huanqin Dai
J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.0c04460 • Publication Date (Web): 25 Aug 2020
Downloaded from pubs.acs.org on August 25, 2020
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Lanostane Triterpenoids with PTP1B Inhibitory and Glucose-Uptake
Stimulatory Activities from Mushroom Fomitopsis pinicola Collected
in North America
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Jinjin Zhang1,2,#, Baosong Chen1,2,#, Jack Liang3, Junjie Han1, Liwei Zhou1, Ruilin
Zhao1, Hongwei Liu 1,2*, and Huanqin Dai1,2,*
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1State Key Laboratory of Mycology, Institute of Microbiology, Chinese Academy of
Sciences, Beijing, P. R. China
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2Savaid Medical School, University of Chinese Academy of Sciences, Beijing, P. R.
China
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3Eastern Health Center, 6801 Mission St, Suite 208, Daly City California, USA
* Corresponding Author Tel: +86 10 64806074; E-mail: liuhw@im.ac.cn (H-W, Liu)
or daihq@im.ac.cn (Huanqin Dai)
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Abstract
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A chemical investigation on the fruiting bodies of Fomitopsis pinicola led to the
isolation and identification of twenty eight lanostane triterpenoids including eleven
new compounds (1-11) and seventeen known analogues (12-28). Their structures
were elucidated by extensive 1D, 2D NMR, and MS spectra. All isolates were tested
for their anti-inflammatory activity, PTP1B inhibitory activity in vitro, and effect on
glucose uptake in insulin-resistant HepG2 cells. Compounds 1, 4, 22, 23 and 27
inhibited the nitric oxide release from the LPS-induced RAW 264.7 cell assay with
IC50 values in the range of 21.4-27.2 μM. Compounds 18, 22, 23, and 28 showed
strong PTP1B inhibitory activity with IC50 values in the range of 20.5-29.9 μM,
comparable to that of the positive control of oleanolic acid (15.0 μM). Compounds 18
and 22 were confirmed to be good competitive inhibitor of PTP1B by kinetic analysis.
In addition, compounds 18, 22, and 28 were found to stimulat glucose uptake in the
insulin-resistant HepG2 cells in the dose from 6.25 to 100 μM. These findings
indicated the potential of F. pinicola in the development of functional food or
medicine for the prevention and treatment of diabetes.
Keywords: Fomitopsis pinicola, lanostane triterpene, anti-inflammatory activity,
PTP1B inhibitory activity, glucose-uptake stimulation
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Introduction
Mushrooms have attracted attention for their production of valuable bioactive
secondary metabolites and their potentials as healthy food and medicinal agents.1-8
Fomitopsis pinicola is a Basidiomycetes mushroom widely distributed in the
coniferous forest all over the world. The fruiting bodies of F. pinicola have been
traditionally used as a functional food and herbal medicine in some Asian countries.9,
10 The extract of F. pinicola was found to show antitumor, antioxidant, hypoglycemic,
and hypolipidemic activities.8,11 Chemical investigations on the fruiting bodies of F.
pinicola have led to the isolation of some steroids and lanostane triterpenoids
possessing bioactivities of antimicrobial effect, cytotoxicity, and inhibitory activity on
COX-1 and COX-2.12-15
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Type II diabetes mellitus (T2DM) is a heterogeneous disease affecting over 100
million people worldwide, which accounts for about 90% of all cases of diabetes.16,17
T2DM is a chronic metabolic disease featured by hyperglycemia and β-cell
dysfunction which was mainly caused by insulin resistance.18-20 Protein tyrosine
phosphatase 1B (PTP1B), a key intracellular non-transmembrane enzyme linked to
T2DM, works as a negative governor for the insulin signaling pathway by
dephosphorylating both the insulin receptor and the downstream insulin receptor
substrate proteins.21-23 Researches in PTP1B knockout rodents have indicated that the
lack of PTP1B gene could improve insulin sensitivity and help prevent diet-induced
obesity without affecting normal growth and longevity.24,25 These findings validated
PTP1B as an attractive therapeutic target for the treatment of type II diabetes. Natural
products with PTP1B inhibitory activity have been discovered in some plants and
fungi.26-32 Examples include styrastilbene B with IC50 of 2.40 μM from Artocarpus
styracifolius, lucidenic acid H and methyl lucidenate F with IC50 of 18.1 and 28.1 μM
from Ganoderma lucidum, clitocybulol L with IC50 of 38.1 μM from Pleurotus
cystidiosus, and erinacerin T with IC50 of 24.9 μM from Hericium erinaceus.26-29
However, there is still an urgent need to develop druggable PTP1B inhibitors with
good cell permeability and specificity.
In an early report, the water extract of Fomitopisis pinicola was demonstrated to
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significantly decrease blood glucose level in diabetic rats induced by streptozotocin.8
However, there is no research on secondary metabolites with hypoglycemic activity
from the fruiting bodies of F. pinicola. In our screening of hypoglycemic activity, the
ethanol extract of F. pinicola showed moderate PTP1B inhibitory activity with IC50 at
about 350 mg/mL. To obtain new mushroom-derived PTP1B inhibitor, a systematic
phytochemical investigation on the ethanol extract of the fruiting bodies of F. pinicola
was carried out. As a result, we isolated and identified eleven new lanostane
triterpenoids (1-11) as well as seventeen known compounds (12-28). All isolated
compounds were assayed for their hypoglycemic activity based on the inhibition on
PTP1B, the stimulation of glucose uptake in insulin-resistance HepG2 cells, and the
anti-inflammatory activity.
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Materials and Methods
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General Experimental Procedures. Optical rotations were obtained on an
Anton Paar MCP 200 automatic polarimeter. IR and UV spectra were recorded on a
Nicolet IS5 FT-IR spectrophotometer and a Thermo Genesys-10S UV−vis
spectrophotometer, respectively. HRESIMS data were recorded on an Agilent
Accurate-Mass-Q-TOF LC/MS 6520 instrument. NMR spectral data were acquired
with a Bruker Avance-500 spectrometer in C5D5N (δH 8.74, 7.58 and 7.22; δC 150.4,
135.9 and 123.9). OD absorbance data were tested on a Spectra Max 190 microplate
reader. Silica gel (Qingdao Haiyang Chemical Co., Ltd.) and octadecylsilyl silica gel
(ODS, 50 μm, YMC CO., LTD) were applied for column chromatography. High
performance liquid chromatography (HPLC) separation was conducted on an Agilent
1200 HPLC system with an reversed phase column (C8, 250×9.4 mm, YMC Pack, 5
μm; detector: UV) with a flow rate of 2.0 mL/min. Analytical grade solvents were
used for extraction and chromatographic separation, and chromatographical grade
solvents were used for HPLC Thin layer chromatography (TLC) analysis was
conducted on silica gel HSGF254 with spots visualized by UV at 254 nm and heated
after spraying with 10% aqueous H2SO4.
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Fungal Material. The fruiting bodies of Fomitopsis pinicola were collected by
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Jack Liang in the Cascade Mountain on the west coast of North America. The
specimen (HMAS255734) was deposited at the Institute of Microbiology, Chinese
Academy of Sciences. This fungus was identified by Dr. Liwei Zhou, one of
co-author.
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Extraction and Isolation. The fruiting bodies of F. pinicola (400g) were crushed
to powder and then extracted with 95% EtOH/H2O (2.5 L) under refluxing at 90°C for
three times, each lasting for 3 h. The combined ethanol extracts were dried under
vacuum to afford the crude extract (113.2 g), which was followed by partition
between water and ethyl acetate. The resulting EtOAc portion (73.6 g) was then
subjected to silica gel column chromatography (CC) using n-hexane-EtOAc in a
gradient elution (v/v, 100:0, 100:1, 80:1, 50:1, 30:1, 20:1, 15:1, 10:1, 5:1), followed
by CH2Cl2-MeOH elution (v/v, 100:0, 100:1, 80:1, 50:1, 30:1, 20:1, 15:1, 10:1, 5:1,
0:100) to give 13 fractions (FP-1~FP-13)(Supplementary ,scheme 2).
Fraction 3 (2.9 g) eluted with n-hexane-EtOAc (v/v, 50:1) was further purified by
RP-HPLC using 80% acetonitrile in water to afford compounds 19 (150.2 mg, tR =
55.0 min), 25 (12.1 mg, tR = 45.3 min), and 28 (300.6 mg, tR = 65.6 min). Fraction 4
(3.0 g) eluted with n-hexane-EtOAc (v/v, 30:1) was purified by RP-HPLC using 70%
acetonitrile in water to give compounds 3 (5.4 mg, tR = 46.0 min), 10 (14.3 mg, tR =
51.5 min), 13 (12.1 mg, tR = 42.8 min), 16 (24.5 mg, tR = 70.6 min), 17 (9.4 mg, tR =
64.5 min), and 26 (12.2 mg, tR = 39.8 min). Fraction 5 (3.9 g) eluted with
n-hexane-EtOAc (v/v, 20:1) was applied on ODS CC eluted with 68% acetonitrile in
water to give nine sub-fractions (FP-5-1~FP-5-9). Compound 9 (4.1 mg, tR = 20.6 min)
was isolated from fraction FP-5-9 (21.5 mg) by RP-HPLC using 60% acetonitrile in
water. Fraction 7 (4.0 g) eluted with CH2Cl2-MeOH (v/v, 80:1) was first separated by
ODS CC using MeOH-H2O in a gradient of 20-80% to give nine sub-fractions
(FP-7-1~FP-7-9). Compound 5 (24.5 mg, tR = 29.1 min) was purified from FP-7-5
(49.0 mg) by RP-HPLC using 50% acetonitrile in water. Fraction FP-7-7 (0.8 g) was
further separated on the silica gel CC with a CH2Cl2-MeOH gradient elution (v/v,
100:0, 100:1, 80:1, 50:1, 30:1, 20:1, 15:1, 10:1, 5:1, 0:100) to give eleven
sub-fractions (FP-7-7-1~FP-7-7-11). Compounds 6 (15.3 mg, tR = 39.8 min), 12 (16.4
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mg, tR = 34.0 min), and 15 (26.3 mg, tR = 30.8 min) were obtained from fraction
FP-7-7-3 by RP-HPLC using 48% acetonitrile in water. Compounds 1 (28.6 mg, tR =
22.4 min) and 7 (32.2 mg, tR = 39.8 min) were isolated from FP-7-7-7 (72.0 mg) by
RP-HPLC using 46% acetonitrile in water. Fraction FP-7-8 (52.8 mg) was
subsequently separated with ODS CC using 68% acetonitrile in water to give eleven
sub-fractions (FP-7-8-1~FP-7-8-11). Compound 14 (4.2 mg, tR = 29.9 min) was
obtained from FP-7-8-8 (5.9 mg) by RP-HPLC using 60% acetonitrile in water.
Fraction FP-7-9 (66.0 mg) was separated on an ODS CC using 65% acetonitrile in
water to give twelve sub-fractions (FP-7-9-1~FP-7-9-12). Compound 11 (10.2 mg, tR
= 21.4 min) was purified from fraction FP-7-9-9 (14.4 mg) by RP-HPLC using 60%
acetonitrile in water. Compound 27 (4.1 mg, tR = 25.3 min) was purified from
FP-7-9-10 (33.5 mg) by RP-HPLC using 56% acetonitrile in water. Compounds 4 (4.3
mg, tR = 31.3 min) and 24 (6.2 mg, tR = 24.5 min) were obtained from fraction
FP-7-9-11 (13.7 mg) by RP-HPLC using 55% acetonitrile in water. Compound 21
(5.2 mg, tR = 25.7 min) was isolated from FP-7-9-12 (7.3 mg) by RP-HPLC using
58% acetonitrile in water. Fraction FP-7-10 (502.0 mg) was subjected to a silica gel
CC with a dichloromethane-methanol gradient elution (v/v, 50:1, 30:1, 20:1, 15:1,
10:1, 5:1, 0:100) to give twelve sub-fractions (FP-7-10-1~FP-7-10-12). Fraction
FP-7-10-4 (52.0 mg) was further separated by ODS CC eluted with 50% acetonitrile
in water to give ten sub-fractions (FP-7-10-4-1~FP-7-10-4-10). Compound 18 (8.2 mg,
tR = 29.9 min) was isolated from FP-7-10-4-7 (13.1 mg) by RP-HPLC using 55%
acetonitrile in water. Compound 23 (4.9 mg, tR = 31.1 min) was isolated from
FP-7-10-4-9 (7.2 mg) by RP-HPLC using 57% acetonitrile in water. Fraction
FP-7-10-5 (60.0 mg) was separated by ODS CC using 55% acetonitrile in water to
give thirteen sub-fractions (FP-7-10-5-1~FP-7-10-5-13). Fraction FP-7-10-5-10 (6.9
mg) was separated by RP-HPLC using 52% acetonitrile in water to afford compound
2 (5.2 mg, tR = 20.9 min). Compound 8 (4.2 mg, tR = 24.3 min) was isolated from
FP-7-10-5-12 (6.6 mg) by RP-HPLC using 50% acetonitrile in water. Compounds 20
(11.3 mg, tR = 27.5 min) and 22 (6.2 mg, tR = 30.1 min) were separated from
FP-7-10-6 (24.4 mg) by RP-HPLC using 57% acetonitrile in water.
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Formipiniate A (1). White powder; [α]25D = +16.0 (c 0.1, MeOH); UV (MeOH)
λmax (log ε): 250 (4.16) nm; IR (neat) νmax: 3424, 2945, 1705, 1674, 1465, 1452, 1216
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cm-1; H and C NMR data, Table 1; HRTOFMS m/z [M+H]+ 541.3525 (calcd. for
C33H49O6, 541.3529).
Formipiniate B (2). White powder; [α]25D = +26.1 (c 0.1, MeOH); UV (MeOH)
λmax (log ε): 246 (4.20) nm; IR (neat) νmax: 3432, 2966, 1724, 1632, 1455, 1247 cm-1;
1H and 13C NMR data, Table 1; HRTOFMS m/z [M+H]+ 527.3733 (calcd. for
C33H51O5, 527.3736).
Forpinic acid D (3). White powder; [α]25D = -13.9 (c 0.1, MeOH); UV (MeOH)
λmax (log ε): 248 (4.34) nm; IR (neat) νmax: 3438, 2955, 1742, 1702, 1646, 1456, 1381,
1266 cm-1; 1H and 13C NMR data, Table 1; HRTOFMS m/z [M+H]+ 481.3315 (calcd.
for C31H45O4, 481.3318).
Forpinic acid E (4). White powder; [α]25D = +26.9 (c 0.1, MeOH); UV (MeOH)
λmax (log ε): 248 (4.22) nm; IR (neat) νmax: 3449, 2966, 1724, 1642, 1383, 1194 cm-1;
1H and 13C NMR data, Table 2; HRTOFMS m/z [M+H]+ 497.3262 (calcd. for
C31H45O5, 497.3267).
Forpinic acid F (5). White powder; [α]25D = -16.1 (c 0.1, MeOH); UV (MeOH)
λmax (log ε): 246 (4.46) nm; IR (neat) νmax: 3426, 2974, 1722, 1663, 1630, 1461, 1384,
1207 cm-1; 1H and 13C NMR data, Table 2; HRTOFMS m/z [M+H]+ 641.4050 (calcd.
for C38H57O8, 641.4053).
Forpinic acid G (6). White powder; [α]25D = +13.2 (c 0.1, MeOH); UV (MeOH)
λmax (log ε): 245 (4.32) nm; IR (neat) νmax: 3442, 2970, 1744, 1709, 1655, 1466, 1385,
1232, 1087 cm-1; H and C NMR data, Table 2; HRTOFMS m/z [M+H]+ 585.3788
1
13
(calcd. for C35H53O7, 585.3791).
Formipiniate C (7). White powder; [α]25D = +57.9 (c 0.1, MeOH); UV (MeOH)
λmax (log ε): 206 (4.46) nm; IR (neat) νmax: 3431, 2928, 1699, 1626, 1459, 1384, 1203
1
13
cm-1; H and C NMR data, Table 3; HRTOFMS m/z [M+H]+ 543.3682 (calcd. for
C33H51O6, 543.3686).
Formipiniate D (8). White powder; [α]25D = +29.9 (c 0.1, MeOH); UV (MeOH)
λmax (log ε): 206 (4.16) nm; IR (neat) νmax: 2969, 1732, 1631, 1462, 1377, 1201 cm-1;
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1H and 13C NMR data, Table 3; HRTOFMS m/z [M+H]+ 541.3890 (calcd. for
C34H53O5, 541.3893).
Formipiniate E (9). White powder; [α]25D = +35.9 (c 0.1, MeOH); UV (MeOH)
λmax (log ε): 202 (4.28) nm; IR (neat) νmax: 3409, 2966, 1724, 1622, 1455, 1381, 1211
1
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cm-1; H and C NMR data, Table 3; HRTOFMS m/z [M+Na]+ 541.3890 (calcd. for
C33H52O5Na, 541.3893).
Formipinic acid G (10). White powder; [α]25D = -8.0 (c 0.1, MeOH); UV (MeOH)
λmax (log ε): 204 (4.19) nm; IR (neat) νmax: 3422, 2960, 1744, 1640, 1455, 1389, 1263
1
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cm-1; H and C NMR data, Table 4; HRTOFMS m/z [M+H]+ 513.3577 (calcd. for
C32H49O5, 513.3580).
Formipinic acid H (11). White powder; [α]25D = -41.9 (c 0.1, MeOH); UV (MeOH)
λmax (log ε): 201 (4.07) nm; IR (neat) νmax: 3397, 2944, 1745, 1638, 1449, 1382, 1204
1
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cm-1; H and C NMR data, Table 4; HRTOFMS m/z [M+Na]+ 651.4051 (calcd. for
C37H56O8Na, 651.4053).
Determination
of
the
Absolute
Configuration
in
the
3-hydroxy-3-methylglutaroyl (HMG) moiety of 16.33 Firstly, 16 (5.0 mg, 8.3 μmol)
was dissolved in 0.5 mL of DMF, (S)-1-Phenylethylamine (1.9 μL, 15 μmol), Et3N
(3.2 μL, 22.5 μmol), HOBt (2.0 mg, 15 μmol) and PyBOP (5.9 mg, 11.5 μmol) were
added to the solution at 0 °C. Then the mixture was stirred at room temperature
overnight and quenched with dilute aqueous HCl, extracted with EtOAc to afford a
yellow residue, which was purified by RP-HPLC using 45% acetonitrile in water to
give amide 16a (3.9 mg). 16a (3.9 mg, 5.5 μmol) was dissolved in 0.5 mL THF and
then LiBH4 (1.8 mg, 82.4 μmol) was added to it under ice-cooling. The mixture stirred
for 24 h at room temperature, then stopped with diluted aqueous HCl, and extracted
with EtOAc. The resulting material was then purified by RP-HPLC using 40%
acetonitrile in water to give a colorless oil, which was further acetylated with Ac2O
(3.2 μL, 29.1 μmol) in pyridine (100 μL). The mixture was stirred for 24 h at room
temperature and then diluted with water, extracted with EtOAc, and concentrated to
1
afford 16b (1.9 mg). The H NMR spectrum of 16b was consistent with that of
(3R)-5-O-acetyl-1-[(S)-phenylethyl]mevalonamide, rather than that of the (3S) isomer
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reported in the literature.33
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(3R)-5-O-Acetyl-1-[(S)-phenylethyl]mevalonamide (16b): colorless oil; [α]25D = -58.0
(c 0.1, CHCl3); 1H NMR (CDCl3, 500MHz) δ 7.27-7.36 (5H, m, C6H5-1’), 6.03 (1H, d,
J = 6.2 Hz, NH), 5.15 (1H, quintet, J = 7.2 Hz, H-1’), 4.24 (2H, td, J = 7.0, 2.9 Hz,
H-5), 2.41 and 2.29 (each 1H, d, J = 14.9 Hz, H-2), 2.04 (3H, s, 5-OCOCH3), 1.86
(2H, m, H-4), 1.51 (3H, d, J = 7.2 Hz, H-2’), 1.24 (3H, s, H-6); positive HRTOFMS
m/z [M+Na]+ 316.1523 (calcd for C16H23NO4Na, 316.1525).
1
(3S)-5-O-Acetyl-1-[(S)-phenylethyl]mevalonamide: colorless oil; H NMR (CDCl3,
500MHz) δ 7.27-7.36 (5H, m, C6H5-1’), 6.03 (1H, d, J = 6.2 Hz, NH), 5.15 (1H,
quintet, J = 7.2 Hz, H-1’), 4.20 (2H, td, J = 7.0, 2.9 Hz, H-5), 2.41 and 2.29 (each 1H,
d, J = 14.9 Hz, H-2), 2.00 (3H, s, 5-OCOCH3), 1.82 (2H, m, H-4), 1.51 (3H, d, J = 7.2
Hz, H-2’), 1.23 (3H, s, H-6)
Anti-inflammatory Activity. The anti-inflammatory activity of compounds was
estimated using LPS-induced RAW 264.7 cells as reported in a literature.34
Cytotoxicity was first evaluated by CCK8 colorimetric assay before the
anti-inflammatory evaluation. Inhibition (%) was calculated and IC50 was determined
graphically as previously reported.34
PTP1B Inhibitory Activity Assay. PTP1B used in the current study was
purchased from LABLEAD Biotechnology Co., Ltd. (Beijing, China). The inhibitory
activity of all isolates against PTP1B was tested in 96-well microplates as previously
reported.35 In brief, 4 mM p-NPP and 100 nM PTP1B were added to each well of a
96-well plate in 200 μL reaction mixture containing 50 mM citrate (pH 6.5), 1 mM
EDTA, 0.1 M NaCl, and 1 mM DTT with or without the test compounds. After
incubation at 37 °C for 30 min, the reaction was stopped by addition of 1 M NaOH.
The absorbance of the reaction mixture was measured at 405 nm using a Spectra max
190 microplate reader (Molecular Devices Inc.). All assays were conducted in
triplicate.
Inhibition Kinetic Assay. Lineweaver-Burk double reciprocal plot and Dixon
plot were used to study the kinetic behavior of active compounds against PTP1B and
the corresponding inhibition constants (Ki values). Enzyme reactions were conducted
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at various concentrations of p-NPP substrate (1, 2, 3 and 4 mM) with active
compounds at different concentrations (0, 6.25, 12.5, and 25 μM for 18, 0, 3.125, 6.25,
and 12.5 μM for 22) and 100 nM PTP1B in 96-well plates. The plate reader recorded
the absorbance of the reaction mixture at every three minutes. The enzymatic velocity
of the enzyme reaction was calculated based on the time-Δabs plot. The
lineweaver-Burk plot and Dixon plot were all generated by GraphPad Prism 5 in order
to determine the type of PTP1B inhibition and Ki. All assays were conducted in
triplicate.
2-NBDG Glucose Uptake Assay. HepG2 Human hepatocyte carcinoma cells
were purchased from National Infrastructure of Cell Line Resource (Beijing, China)
and cultured in Dulbecco’s modified eagle medium (DMEM).36-39 Glucose uptake
37
activity was tested as previously reported with minor changes . Cells were cultured
in 96-well plates and treated with 10−6ꢀM insulin for 24ꢀh in serum-free DMEM to
induce insulin resistance. Serial dilutions of the compounds from 100 μM were added
and incubated for 24 h, then 100 nM insulin was added for a 30 min incubation at
37oC. After that, 50 μM 2-NBDG was added for 30 min, and cells were subsequently
washed with PBS. 2-NBDG uptake was measured on a microplate reader at 485 nm
excitation and 528 nm emission. DMSO and rosiglitazone were used as negative and
positive controls. Five replicate wells were established and each experiment was
repeated three times. Relative fluorescence intensity minus background was used for
statistical analyses. All data were calculated by GraphPad Prism 5 and presented as
mean ± SD of three independent experiments.
Molecular Docking Analysis. The crystal structure of PTP1B with an inhibitor
{[5-(3-{[1-(benzylsulfonyl)piperidin-4-yl]
amino}
phenyl)
-4-bromo-2-(2H-tetrazol-5-yl)-3-thienyl]oxy}acetic acid (PDB ID: 2NT7) was
obtained from the RCSB Protein Data Bank (PDB) database. The original inhibitor
and water molecules were deleted from the crystal structure before docking
simulation using PyMOL (version 1.0.0.0) and the protein was saved in PDB format
(receptor.pdb). The ligand structures were first drawn using ChemDraw 2D software
(version 14.0) and converted to 3D structures by ChemDraw 3D software, then the 3D
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structures were subjected to energy minimization using the MM2 menu in ChemDraw
3D and deposited as files in PDB format (ligand.pdb). Both of the receptor.pdb and
ligand.pdb were transformed to PDBQT formate by AutoDockTools (version 1.5.6)
for the subsequent docking. Docking simulation was conducted using Autodock Vina
40 and the docking calculation results were analyzed using PyMOL.
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293
294
295
296
297
298
Results and Discussion
The ethanol extract of F. pinicola was partitioned between water and ethyl acetate.
299
300
301
302
303
304
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306
307
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309
310
311
312
313
314
315
316
317
318
319
320
The ethyl acetate-soluble portion was further separated by series of column
chromatographic including silica gel, ODS, and preparative HPLC to give
twenty-eight lanostane triterpenoids, including eleven new compounds (1-11) and
seventeen known analogues (12-28) (Figure 1). Compounds 12-28 were identified to
be 16α-hydroxy-3-oxolanosta-7,9(11),24-trien-21-oic acid (12),41 dehydroeburicoic
acid (13),42 16α-acetyloxy-24-methylene-3-oxo-5α-lanost-7,9(11)-dien-21-oic acid
(14),12
polyporenic
acid
C
(15),12
formipinic
acid
A
(16),15
3β-hydroxy-5α-lanosta-8,24-dien-21-oic acid (17),43 formipinic acid D (18),15
3-oxo-5α-lanosta-8,24-dien-21-oic acid (19),43 21-hydroxylanosta-8,24-dien-3-one
(20),12
palustrisoic
acid
F
(21),44
3α-(3’-hydroxy-4’-carboxyl-3’-methyl-1,5-dioxopentyloxy)-24-methylene-5α-lanost-
8-en-21-oic acid(22),45 polyporenic acid D (23),46 polyporenic acid A (24),47
3β-acetyloxy-16α-hydroxylanosta-8,24(31)-dien-21-oic
acid
(25),48
16α-acetoxy-24-methylene-3-oxolanost-8-en-21-oic acid (26),49 dehydroeburiconic
acid (27),50 and pinicolic acid C (28)51 by comparing their NMR and MS spectroscopic
data with the literature data. The structure elucidations of new compounds were
described as follows:
The molecular formula of formitopsic acid C (1) was determined to be C33H48O6
(10 degrees of unsaturation) on the basis of HRTOFMS data ([M+H]+, m/z 541.3525;
1
calcd. 541.3529). Analysis of its H, 13C NMR and HSQC spectra revealed the
characteristic resonances for a 7, 9(11)-diene lanostane-type triterpene (Table 1).33
The HMBC correlations from H3-18 to C-12, C-13, C-14 and C-17, from H3-19 to
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338
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341
342
343
344
345
346
347
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349
350
C-1, C-5, C-9 and C-10, from H3-28 and H3-29 to C-3 (δC 215.7), C-4, and C-5, from
H3-30 to C-8, C-13, C-14, and C-15 [δH/δC 4.70 (dd, J = 6.0, 9.4 Hz)/74.0], from H-17
(δH 1.69 overlapped) to C-21 (δC 66.1), from H2-21 [δH 4.34 (dd, J =5.6, 11.4 Hz);
4.63 (dd, J = 3.1, 11.4 Hz)] to C-20 and C-22, from H3-26 and H3-27 to C-24 and
1
C-25, as well as the H-1H COSY correlations indicated in Figure 2 assigned the
lanostane skeleton and confirmed the presence of ketone at C-3, hydroxyl group at
C-15, and oxygenated methylene at C-21, respectively. The remaining carbonyl
groups at δC 168.7 (C-1’) and 170.2 (C-3’) and methylene at δH/δC 3.90 (s)/43.4 were
assembled into a malonic acid moiety attached at C-21 via an ester bond, as deduced
from the HMBC correlations of H2-2’ with C-1’ and C-3’，and H2-21 with C-1’. The
relative configurations of 1 were established by a ROESY experiment. Significant
NOE correlations of H3-28 with H-5, H-5 with H3-30, H3-30 with H-17, H3-29 with
H3-19, H3-19 with H3-18 and H3-18 with H-15 indicated α configuration for H-5,
H-17 and H3-30, β configuration for H-15, H3-18, H3-19. NOE correlation of H3-18
with H2-20 combining with the biosynthetic pathway of lanostane triterpenoids
reported in the literature,52 the relative configuration of H2-20 was determined as β.
Thus,
compound
1
was
determined
to
be
3-oxo-21-carbonylpropinonyl-15α-hydroxy-lanosta-7, 9(11), 24-trien.
The molecular formula of formitopsic acid D (2) was established as C33H50O5 by
1
HRTOFMS data ([M+H]+, m/z 527.3733; calcd. 527.3736). A comparison of H and
13C NMR spectroscopic data between 1 and 2 indicated the loss of a carbonyl carbon
and the presence of an extra oxygenated methine. The location of the hydroxy group
p
1
2
(
H
3
2
(
H
δ
2
2
4
(1
5
4
(
5
at C-3 was confirmed by HMBC correlations from H-3 [δH3.49 (dd, J = 9.0, 6.5 Hz)]
T
3
1
1
1
5
to C-1, C-2, and C-4, from H3-28 [δH 1.24 (s)] and H3-29 [δH 1.16 (s)] to C-3 (δC 78.6)
and 1H-1H COSY correlations of H2-1/H2-2/H-3. The structure of 2 was fully assigned
by detailed analysis of 2D NMR data. The relative configuration of H-3 was
established to be α based on NOE correlations of H-3 with H-5 and H3-28, H3-29 with
H-19, in combination with the splitting pattern and coupling constant analysis of H-3
[δH 3.49 (dd, J = 9.0, 6.5 Hz)]. Research showed when H-3 was β-oriented, H-3 was a
broad singlet, whereas, H-3α corresponded to a dd (J=5-7 and 9-12 Hz) or t peak
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(J=7-8
Hz).53
Thus,
compound
2
was
determined
to
be
352
353
354
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356
357
358
359
360
361
362
363
364
365
366
367
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369
370
371
372
373
374
375
376
377
378
379
380
3β-hydroxyl-21-carbonylpropinonyl-lanosta-7, 9(11), 24-dien.
Forpinic acid D (3) possessed a molecular formula of C31H44O4, as determined by
HRTOFMS data ([M+H]+, m/z 481.3315; calcd. 481.3318). Its H, 13C NMR and
1
HSQC spectra showed the presence of seven methyls, eight methylenes including one
sp2 methylene [δH/δC 4.91 (s), 5.05 (s)/107.0], six methines including two sp2
methines [δH/δC 5.41 (d, J = 6.2 Hz)/117.3, 5.51 (d, J = 6.5)/121.9], and ten quaternary
carbons including three sp2 carbons (δC 139.9, 144.8, 155.4), and three carbonyl
carbons (δC 214.7, 216.9, 177.0). A comparison of NMR data between 3 and
polyporenic
acid
C
(15)12
revealed
a
skeleton
of
3-oxo-24-methylene-lanosta-7,9(11),24(31)-trien-21-oic acid. The substitution of a
ketone at C-16 in 3 instead of a hydroxyl group in 15 was confirmed by the splitting
pattern of H2-15 [δH 2.27 (d, J = 17.9 Hz), 2.57 (d, J = 18.1 Hz)] and the HMBC
correlations from H2-15 to C-16 (δC 216.9), from H-17 [δH 3.07 (m)] and H-20 [δH
2.99 (m)] to C-16. Thus, the planar structure of compound 3 was assigned as 3,
16-dioxo-24-methylene-lanosta-7, 9(11), 24(31)-trien-21-oic acid.
Forpinic acid E (4) had a molecular formula of C31H44O5, as determined by
HRTOFMS data ([M+H]+, m/z 497.3262; calcd. 497.3267). Its H and 13C NMR
1
spectroscopic data were similar to those of 3 except for the presence of an oxygenated
methine [δH/δC 4.65 (d, J = 10.2 Hz)/66.8] and the loss of one methylene. The
oxygenated methine was assigned to be C-6 on the basis of the HMBC correlations
from H-6 to C-4 (δC 47.3), C-5 (δC 56.0), C-7 (δC 129.1) and C-8 (δC 138.0), in
1
combination with the H-1H COSY correlations of H-5/H-6/H-7. The structure of 4
was fully assigned by detailed analysis of the 2D NMR data. The relative
configuration of H-6 was assigned as β by the NOE correlation of H-6 with H3-19 and
H3-29.
The molecular formula of forpinic acid F (5) was determined to be C38H56O8 by
HRTOFMS
data
([M+H]+,
m/z
641.4050;
calcd.
641.4053).
A
3-hydroxyl-24-methylene-lanosta-7,9(11),24(31)-trien-21-oic acid skeleton was
deduced for 5 by comparing its 1H and 13C NMR data with those of dehydroeburicoic
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401
402
403
404
405
406
407
408
409
410
acid (13). The remaining signals including two methylenes [δH/C 3.00
(overlapped)/46.9; 3.06 (overlapped)/46.5], one methyl group [δH/δC 1.67 (s)/28.6],
one methoxyl group [δH/δC 3.61 (s)/51.7], one oxygenated quaternary carbon (δC 70.3),
and two carbonyl carbons (δC 171.6 and 172.4) were assembled into a
3-hydroxy-3-methylglutaroyl (HMG) methyl ester by HMBC spectral analysis.
Furthermore, the HMBC correlations from H-3 to C-1’, from H2-15, H-17, and H-20
to C-16 (δC 217.7) confirmed the substitution of a HMG moiety at C-3 via an ester
bond and the presence of a ketone at C-16 in 5. The relative configuration of H-3 was
determined to be β based on the splitting pattern of H-3 and the NOE correlation of
H-3 with H3-29.
The molecular formula of forpinic acid G (6) was assigned as C35H52O7 on the
basis of HRTOFMS data ([M+H]+, m/z 585.3788; calcd. 585.3791). Analysis of its 1H
and
13C-NMR
data
showed
a
structural
skeleton
of
24-methylene-lanosta-7,9(11),24(31)-trien-21-oic acid and two acetoxyl groups [δH/δC
1.89 (s)/21.1; 2.04 (s)/21.5; 170.8; 171.1]. The presence of acetoxyl groups at C-3 and
C-28 and the substitution of a hydroxyl moiety at C-16 were indicated by the key
HMBC correlations from H-3 [δH 5.09 (dd, J = 11.8, 4.2 Hz)] to an ester carbonyl (δC
170.8), C-4 (δC 41.5) and C-28 (δC 66.1), from H2-28 [δH 4.10 (d, J = 11.6 Hz); 3.99 (d,
J = 11.6 Hz)] to C-4, C-5 (δC 43.3), C-29 (δC 13.9), and an ester carbonyl (δC 171.1),
and from H-16 [δH 4.53 (t, J = 7.1 Hz)] to C-15 (δC 44.8), C-17 (δC 58.0), and C-20 (δC
48.9). Finally, the structure of 6 was fully assigned by comprehensive analysis of the
1H-1H COSY, HSQC, and HMBC spectral data. The relative configuration of 6 was
solved by ROESY experiment. The NOE correlations of H-3 with H-5 and H2-28,
H-16 with H-20 and H3-19 supported the α configuration of H-3 and β-orientation of
H-16.
A molecular formula of C33H50O6 was assigned for formitopsic acid E (7) based
on the HRTOFMS data ([M+H]+, m/z 543.3682; calcd 543.3686). The 1D NMR
spectroscopic data of 7 were similar to those of 1 except for the loss of two protonated
olefinic carbons in 7. The HMBC correlations from H2-7 [δH 2.57 (m)] to C-8 and C-9,
from H2-11 [δH 1.98 (m)] to C-8 and C-9, from H3-19 [δH 1.09 (s)] to C-9 (δC 134.1),
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and from H3-30 [δH 1.31 (s)] to C-8 (δC 136.2) confirmed the presence of a double
bond between C-8 and C-9. The α configuration of 15-OH was indicated by NOE
correlation of H-15 with H3-18. The structure of 7 was assigned as
3-oxo-21-carbonylpropinonyl-15α-hydroxy-lanosta-8, 24-dien.
412
413
414
415
416
417
418
419
420
421
422
423
424
425
426
427
428
429
430
431
432
433
434
435
436
437
438
439
440
The molecular formula of formipiniate A (8) was determined to be C34H52O5
from the HRESIMS data at m/z 541.3890 ([M+H]+, calcd. 541.3893). A detailed
comparison of 1D NMR between 8 and 7 showed the presence of one additional
methoxyl group and the absence of an oxygenated methine in 8. On the basis of the
HMBC correlation from the methoxyl group [δH/δC 3.72 (s)/52.8] to one carbonyl
carbon (δC 168.0), a methyl ester of malonic acid was deduced in 8. With the
assistance of 2D NMR data, the structure of 8 was finally determined to be
3-oxo-21-(3’-methoxylcarbonylpropinonyl)-lanosta-8, 24-dien.
Formitopsic acid F (9) had a molecular formula of C33H52O5, as determined by
1
13
HRTOFMS ([M+H]+, m/z 529.3890; calcd. 529.3893). Its H and C NMR spectra
resemble those of 8, except for the loss of the carbonyl moiety and the methoxyl
group. The location of a hydroxyl group at C-3 was confirmed by HMBC correlations
from H-3 [δH 3.48 (t, J = 9.7 Hz)] to C-4 (δC 40.0), from H3-28 [δH 1.26 (s)] and H3-29
1
[δH 1.10 (s)] to C-3 (δC 78.6), and H-1H COSY correlations of H2-1/H2-2/H-3. The
structure of 9 was fully assigned by detailed analysis of 2D NMR data. The 3β-OH
was supported by the splitting pattern and coupling constant of H-3 as well as the
NOE correlations of H-3 with H-5 and H3-28. Therefore, the structure of 9 was
established as 3β-hydroxy-21-carbonylpropinonyl-lanosta-8, 24-dien.
The structure of formipinic acid G (10) and H (11) was assigned by analysis of
the HRTOFMS, 1D and 2D NMR data, respectivley. Compound 10 is determined to
be an epimer of formipinic acid D (18),15 which was indicated by the splitting pattern
and coupling constant of H-3 [δH 4.64 (dd, J = 11.8, 4.5 Hz)] rather than a broad
singlet at δH 4.87 in 18. Moreover, the NOE correlations of H-3 with H-5 and H3-28
supported the substitution of 3β-acetoxyl group at C-3. The structure of 11 differs
13
from that of 10 in the substitution group at C-3. The C NMR spectra of 11 showed
diagnostic resonances of a 3-hydroxy-3-methylglutaroyl (HMG) methyl ester (δH/δC
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442
443
444
445
446
447
448
449
450
451
452
453
454
455
456
457
458
459
460
461
462
463
464
465
466
467
468
469
470
171.7, 3.06 (overlapped)/46.9, 70.3, 1.69 (s)/28.9, 3.04 (overlapped)/46.5, 172.4, 3.62
(s)/51.8)15 that was also supported by HMBC correlations of H-2’ with C-1’ (δC 171.7),
H-5’ with C-6’ (δC 172.4) and of 6’-OCH3 with C-6’. The HMBC correlations from
H-3 [δH 4.95 (brs)] to C-1’ (δC 171.7,) indicated that the linkage of HMG group to C-3
via an ester bond. The relative configuration of H-3 was established to be β based on
the NOE correlations of H-3 with H3-19.
To determine the absolute configuration of C-3’ in the HMG moiety of 5, 11, 16,
21 and 22, a chemical derivatization method33 was applied on 16 that was obtained as
one of major compound. Compound 16a was obtained by amidation of 16 with
(S)-1-phenylethylamine. Then 16a was reducted with LiBH4, and acetylated by Ac2O,
1
gave 5-O-acetyl-1-[(S)-phenylethyl]mevalonamide (16b), which had the same H
NMR data as those of (3R)-5-O-acetyl-1-[(S)-phenylethyl]mevalonamide. As a result,
the absolute configuration of C-3’ in the HMG group of compound 16 was
determined as S. Considering the similar 1D NMR spectroscopic data of the HMG
moiety in 5, 11, 16, 21, and 22 and the same mevalonate biosynthetic origin of this
moiety, we deduced S-configuration for the HMG moiety in 5, 11, 21, and 22.
Evaluation of Biological Activities. All isolates were assayed for their
anti-inflammatory activity, PTP1B inhibitory activity, and the stimulatory effect on
glucose uptake in insulin-resistant HepG2 cells.
Anti-inflammatory Activity. Compounds 1, 3-18, 20-25, 27 and 28 that had no
cytotoxity on RAW 264.7 cells at the dose of 100 μM were assayed for their
anti-inflammatory activity by evaluating the NO (nitric oxide) production in
LPS-induced RAW 264.7 cells (Table 5). As a result, compounds 1, 4, 22, 23 and 27
were found to decrease the NO release in LPS-induced RAW 264.7 cells with IC50
values in the range of 21.4-27.2 μM. Other compounds showed weak inhibitory
activity with IC50 values larger than 100 μM.
PTP1B Inhibitory Activity of Compounds 1-28. All compounds were also evaluated
for their abilities to inhibit PTP1B activity in vitro. As shown in Table 6, Compounds
5, 7, 18, 22, 23 and 28 showed PTP1B inhibition with IC50 values in the range of
20.5-56.4 μM, while other compounds were weak or inactive. Compounds 18, 22, 23,
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and 28 exhibited better inhibition activities with IC50 values of 21.5, 20.5, 25.9, 29.9
μM, respectively. The preliminary structure-activity relationship was analyzed for
compounds 1-28 on the basis of the PTP1B inhibitory activity. It was found that the
substitution at C-3 and the double bond between C-8 and C-9 in the lanostane
triterpenes might play a vital role in inhibiting PTP1B. The 3-carbonyl group may
lead to relatively weak activity. However, when C-3 was substituted by the HMG
group, it could lead to a stronger PTP1B inhibitory activity. For example, compound
22 had the same structure as 27, with the only difference in the replacement of
carbonyl group by α-HMG in 22. Compound 22 showed relatively strong PTP1B
inhibitory activity, while compound 27 had no activity. It is the same for compounds
3 and 5. In addition, the relative configuration of the substitution group at C-3
contributes much to PTP1B activity. Compound 10 had the same planar structure as
compound 18, with the only difference in the relative configuration of 3-OAc which
in 10 is β but in 18 is α. Compound 18 showed obvious PTP1B inhibitory activity;
nevertheless, compound 10 is inactive. Furthermore, compound 7 possessing a double
bond between C-8 and C-9 displayed stronger activities in stimulating the PTP1B
inhibition than compound 1, which has a 7, 9 (11)-dien moiety.
472
473
474
475
476
477
478
479
480
481
482
483
484
485
486
487
488
489
490
491
492
493
494
495
496
497
498
499
500
Kinetic Characterization. Lineweaver-Burk double reciprocal plot and Dixon plot
were further applied to study the kinetic behavior of compounds 18 and 22 against
PTP1B. As shown in Figure 4A and 4B, the maximum rate of the PTP1B-catalyzed
reaction was not changed, and the Km was increased with the increase of
concentrations. Therefore, compounds 18 and 22 are competitive inhibitors against
PTP1B. In the Dixon plot method (Figure 4C and 4D), the Ki values of 18 and 22
were determined to be 20.05 μM and 9.34 μM, relatively. The relatively lower Ki
value of 22 makes it to be a promising competitive PTP1B inhibitor.
Molecular Docking Analysis. To gain insight into the interaction and binding mode
between the active compounds 18, 22, 23, 28, oleanolic acid and PTP1B, molecular
docking simulation was performed using AutoDock Vina. As indicated in Figure 5A,
compounds 18, 22, 23, 28 can dock into the same active cavity as that of oleanolic
acid with the low binding energy (18: -6.80 kcal/mol; 22: -7.60 kcal/mol; 23: -7.20
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501
502
503
504
505
506
507
508
509
510
511
512
513
514
515
516
517
518
519
520
521
522
523
524
525
526
527
528
529
530
kcal/mol; 28: -6.10 kcal/mol; oleanolic acid: -7.40 kcal/mol). The H-bond interactions
between the carboxylic acid of 18, 22, 23, 28, oleanolic acid and the indicated amino
acid residues Y20, R24, H25, A27, S28, F182, R221, R254, G259, Q262, and Q266 in
the catalytic site of PTP1B were shown in Figure 5B-5F. These results were
consistent with the earlier discovery that several PTP1B inhibitors could interacted
with the enzyme site surrounding the catalytic amino acid Cys215 and the
neighboring area.54
Glucose Uptake-Stimulating of Compounds 18, 22, 23, and 28. Lanostane
37
triterpenoids from Wolfiporia cocos showed glucose uptake utimulatory activity.
Encouraged by the result of PTP1B inhibition assay, compounds 18, 22, 23, and 28
were tested for their effects on glucose uptake in insulin-resistant HepG2 cells. All
tested compounds showed no cytotoxicity against HepG2 cells at the dose up to 100
μM. As shown in figure 6, compounds 18, 22, 28 enhanced the insulin-stimulated
uptake of 2-NBDG in insulin-resistant HepG2 cells within the concentration range of
(100-6.25 μM). At the dose of 12.5 μM, compounds 18, 22 and 28 enhanced the
insulin-stimulated uptake of 2-NBDG by 44.6 %, 31.5 %, and 15.3 %, respectively,
which were similar to that of the positive control of rosiglitazone (40.2%). Compound
23 showed little effect on glucose uptake.
Many studies have shown that PTP1B is related to diabetes, obesity, cancer and
inflammation. PTP1B inhibitors could improve insulin sensitivity of tissues by
preventing the dephosphorylation of insulin receptors and the downstream insulin
receptor substrates, regulate the insulin pathway positively and as a result, stimulate
the glucose uptake and ameliorate glucose metabolism disorders. PTP1B was also
reported to involve in the development of inflammation.55 Overexpression of PTP1B
could enhance the activation of macrophages and microglia induced by LPS and
promote the release of inflammatory factors. Therefore, compounds with good PTP1B
inhibition activities may not only improve glucose metabolism, but also could develop
as potential anti-inflammatory drugs.
Conclusions. Eleven new lanostane triterpenoids (1-11) as well as seventeen known
lanostane triterpenoids (12-28) were identified from the fruiting bodies of F. pinicola
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collected in North America. Compounds 1, 4, 22, 23 and 27 inhibited the release of
NO from LPS-stimulated macrophage, showing good anti-inflammtory activity.
Compounds 18, 22, and 28 exhibited both strong PTP1B inhibitory activity in vitro
and the promising glucose uptake stimulating activity in insulin-resistant HepG2 cells.
The molecular docking and kinetic analysis further confirmed the interaction between
compounds 18, 22, 23, and 28 and PTP1B. The bioactive lanostane triterpenoids from
F. pinicola deserve further investigation for their action mechanisms. Our current
findings provide new evidence for the development and utilization of the edible and
medicinal F. pinicola in the prevention and treatment of diabetes.
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540
541
542
Conflicts of Interest
There are no conflicts to declare.
Acknowledgements
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This work was supported by the National Key R&D Program of China (grant No.
2018YFD0400203), the National Natural Science Foundation of China (grant No.
81773614).
Supporting information
1D, 2D NMR, HRTOFMS data, HPLC purity analysis, IR, UV and CD (D) spectra of
compounds 1-11 in Figures S1-S88. Determination of the absolute configuration of
1
HMG group in 16 and the H NMR spectrum of compound 16b in Scheme 1 and
Figure S89. Extraction and isolation procedure of Fomitopsis pinicola in Scheme 2.
HPLC analysis and comparison of crude extract、EtOAc fraction and water fraction
in Figures S90 and S91. The plot curves for IC50 calculation of NO production and
PTP1B inhibitory activities in Figure S92-S93.
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739
740
741
742
743
744
745
746
747
Table 1. The 1H (500MHz) and 13C NMR (125MHz) data of 1-3 in C5D5N
1
2
3
position
1
δC
δH mult.( J in Hz)
2.23 m
δC
δH mult. (J in Hz)
2.04 m
δC
δH mult. (J in Hz)
2.11 m
37.4
36.8
36.2
1.71 m
1.51 m
1.64 m
2
35.5
2.80 m
29.3
1.98 m
34.6
2.75 m
2.40 m
2.37 m
3
4
5
6
215.7
47.9
51.5
24.3
78.6
39.8
50.2
24.0
3.49 dd (9.0, 6.5)
214.7
47.2
50.6
23.6
1.69 overlapped
2.16 m
1.30 dd (11.3, 4.5)
2.18 m
1.60 overlapped
2.12 m
2.05 m
1.99 m
7
8
122.2
142.4
145.7
38.0
6.46 d (6.7)
121.6
143.2
146.9
38.3
5.59 d (6.1)
121.9
139.9
144.8
37.3
5.51 d (6.5)
9
10
11
12
117.7
38.5
5.36 d (6.2)
2.61 m
117.0
37.7
5.36 m
2.44 m
2.12 m
117.3
35.2
5.41 d (6.2)
2.48 m
2.12 m
13
14
15
53.1
44.8
74.0
51.2
44.4
32.3
44.2
42.7
46.5
4.70 dd (9.4, 6.0)
2.23 overlapped
1.76 m
2.57 d (17.9)
2.27 d (17.9)
1.49 m
16
40.6
28.3
2.10 m
216.9
1.43 m
17
18
19
20
21
44.3
17.1
22.6
40.6
66.1
2.34 m
46.1
16.6
23.5
40.7
66.1
2.13 m
57.4
16.6
21.8
44.9
177.0
3.07 m
1.07 s
1.11 s
2.99 m
0.86 s
0.73 s
1.21 s
1.12 s
1.83 m
1.83 m
4.63 dd (11.4, 3.1)
4.34 dd (11.4, 5.6)
1.65 m
4.64 dd (11.4, 3.2)
4.34 dd (11.4, 5.7)
1.82 m
22
23
31.2
25.6
31.1
25.6
30.1
33.9
3.18 m
2.22 m
2.57 m
2.40 m
1.61 m
2.25 m
2.26 m
2.15 m
2.10 m
24
25
125.8
131.8
5.24 t (7.2)
125.9
131.8
5.27 (overlapped)
155.4
32.5
2.32 m
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Journal of Agricultural and Food Chemistry
26
27
28
29
30
31
18.2
26.3
26.1
22.8
18.4
1.63 s
1.67 s
1.13 s
1.08 s
1.34 s
26.3
18.2
29.2
17.1
26.3
1.69 s
1.67 s
1.24 s
1.16 s
1.01 s
21.8
21.7
25.4
22.1
25.3
107.0
1.06 overlapped
1.03 overlapped
1.16 s
1.08 s
1.14 s
5.05 s
4.91 s
1’
2’
3’
168.7
43.4
168.7
43.5
3.90 s
3.89 s
170.2
170.2
748
749
750
751
752
753
Table 2. The 1H (500MHz) and 13C NMR (125MHz) data of 4-6 in C5D5N
4
5
6
position
1
δC
δH mult. (J in Hz)
2.05 m
δC
δH mult. (J in Hz)
1.84 m
δC
35.7
δH mult. (J in Hz)
35.2
31.3
1.90 m
1.88 m
1.67 m
1.47 m
2
30.1
3.15 m
23.6
2.05 m
24.5
1.86 m
2.20 m
1.88 m
1.77 m
3
4
5
6
216.6
47.3
56.0
66.8
78.6
37.2
45.2
23.8
4.95 brs
74.9
41.5
43.3
23.3
5.09 dd (11.8, 4.2)
2.04 d (10.3)
4.65 d (10.2)
1.75 dd (11.2, 4.5)
2.06 m
1.79 dd (11.3, 4.2)
2.16 m
2.12 m
7
8
129.1 5.87 s
138.0
122.7
140.5
146.8
38.2
5.51 d (6.0)
120.8
143.3
146.0
37.9
5.58 d (6.2)
9
143.6
10
11
12
37.4
118.9 5.49 d (6.2)
116.9
35.8
5.44 d (6.4)
2.71 d (17.7)
2.50 m
117.7
36.7
5.35 d (6.4)
2.69 m
35.3
2.76 d (18.1)
2.50 m
2.42 m
13
14
15
43.9
42.8
46.3
45.0
43.5
47.2
49.9
45.5
44.8
2.65 d (17.9)
2.24 d (17.9)
2.55 d (18.2)
2.26 d (18.2)
2.44 overlapped
1.89 overlapped
4.53 t (7.1)
16
17
18
19
20
21
22
216.6
57.3
16.6
23.4
44.8
176.8
29.8
217.7
58.1
17.3
28.9
45.6
177.6
30.9
76.8
58.0
18.1
23.9
48.9
179.1
31.9
3.05 d (10.5)
1.07 s
3.04 overlapped
1.07 s
2.88 dd (11.3, 5.9)
1.06 s
1.05 s
0.99 s
1.05 s
3.00 td (10.5, 3.1)
2.98 m
2.96 td (10.8, 3.1)
1.27 m
2.42 m
2.20 m
2.67 m
2.47 m
2.54 m
2.40 m
23
32.5
33.3
2.48 m
2.40 m
33.7
24
25
26
27
155.4
33.9
21.7
21.6
156.1
34.6
23.1
22.9
156.5
34.6
22.5
22.3
2.34 m
2.34 m
2.29 m
1.04 d (7.2)
1.06 d (7.2)
1.05 d (7.2)
1.04 d (7.2)
1.00 d (6.8)
1.02 d (6.8)
27
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Journal of Agricultural and Food Chemistry
Page 28 of 38
28
30.5
1.72 s
22.5
0.98 s
66.1
4.10 d (11.6)
3.99 d (11.6)
0.98 s
29
30
31
21.8
24.9
1.68 s
1.12 s
22.4
26.4
107.7
0.93 s
1.11 s
5.04 s
4.90 s
13.9
26.9
107.5
1.49 s
107.0 5.05 s
4.92 s
4.99 s
4.86 s
1’
171.6
46.9
70.3
28.6
46.5
172.4
51.7
2’
3’
3.00 overlapped
4’
1.67 s
5’
3.06 overlapped
6’
6’-OCH3
3-OCOCH3
3-OCOCH3
28-OCOCH3
28-OCOCH3
3.61 s
170.8
21.5
2.04 s
1.89 s
171.1
21.1
754
755
Table 3. The 1H (500MHz) and 13C NMR (125MHz) data of 7-9 in C5D5N
7
8
9
position
1
δC
δH (mult. J in Hz)
1.82 m
δC
δH (mult. J in Hz)
1.84 m
δC
36.3
δH (mult. J in Hz)
1.69 m
36.7
36.6
1.51 m
1.49 m
1.19 m
2
35.2
2.62 m
35.2
2.60 m
31.1
1.91 m
2.46 m
2.46 m
1.72 m
3
4
5
6
216.8
47.8
51.7
20.3
216.7
47.9
51.7
20.1
78.6
40.0
51.4
19.2
3.48 t (9.7)
1.65 overlapped
1.60 m
1.61 overlapped
1.55 m
1.20 overlapped
1.74 m
1.55 m
1.50 m
1.56 m
7
8
27.9
136.2
134.1
37.7
21.7
31.9
2.57 m
27.0
135.6
134.2
37.5
21.7
31.1
2.03 m
21.8
134.7
135.7
37.9
29.1
31.2
2.01 m
9
10
11
12
1.98 m
2.07 m
1.66 m
1.95 m
1.85 m
1.60 m
2.03 m
1.92 m
1.68 m
13
14
15
53.0
45.4
72.8
50.7
45.0
31.6
50.7
45.1
28.5
4.57 dd (8.9, 6.1)
2.20 m
1.65 m
2.01 m
1.44 m
2.00 m
1.25 m
16
40.2
28.5
1.99 m
29.2
1.44 m
17
18
19
20
21
44.7
17.3
19.1
40.8
66.2
2.27 m
45.7
16.7
19.2
40.8
66.4
1.96 m
45.8
16.7
19.9
40.8
66.3
2.02 m
0.93 s
0.80 s
0.83 s
1.09 s
1.07 s
1.07 s
1.83 m
1.79 m
1.84 m
4.64 dd (11.4, 3.3)
4.34 dd (11.4, 5.9)
1.68 m
4.58 dd (11.3, 3.2)
4.30 dd (11.3, 5.7)
1.66 m
4.65 dd (11.3, 3.3)
4.34 dd (11.3, 6.0)
1.71 m
22
23
24
31.2
25.6
125.9
31.0
25.5
125.8
31.1
25.5
126.0
1.59 m
1.55 m
1.23 m
2.24 m
2.20 m
2.24 m
2.13 m
2.10 m
2.14 m
5.24 t (7.2)
5.27 t (7.2)
5.27 t (7.3)
28
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Journal of Agricultural and Food Chemistry
25
26
131.7
18.2
26.3
26.9
21.8
18.5
168.8
43.5
170.3
131.8
18.2
26.3
25.8
21.8
24.9
167.7
42.3
168.0
52.8
131.7
1.64 s
1.67 s
1.14 s
1.08 s
1.31 s
1.68 s
1.72 s
1.17 s
1.08 s
0.98 s
18.2
26.3
29.2
16.9
24.9
168.8
43.6
170.5
1.67 s
1.70 s
1.26 s
1.10 s
0.97 s
27
28
29
30
1’
2’
3.89 s
3.81 s
3.72 s
3.88 s
3’
3’-OCH3
756
757
758
759
760
761
Table 4. The 1H (500MHz) and 13C NMR (125MHz) data of 10-11 in C5D5N
10
11
position
1
δC
δH (mult. J in Hz)
1.49 m
δC
δH (mult. J in Hz)
1.64 m
35.5
31.4
1.08 m
1.39 m
2
24.8
1.68 m
27.1
2.01 m
1.56 m
1.94 m
3
4
5
6
81.0
38.5
51.0
18.7
4.64 dd (11.8, 4.5)
78.6
37.4
46.2
18.6
4.95 brs
1.11 overlapped
1.60 m
1.73 overlapped
1.59 m
1.42 m
1.47 m
7
27.4
1.94 m
23.9
1.84 m
1.76 m
8
9
132.6
136.3
37.7
132.4
136.7
37.8
10
11
20.9
1.90 m
20.9
2.04 m
1.98 m
2.20 m
2.06 m
12
29.5
2.21 m
2.02 m
29.5
13
14
15
45.0
44.2
47.2
44.9
44.2
47.1
2.52 d (17.9)
2.04 m
2.56 d (17.8)
2.03 d (17.8)
16
218.4
218.4
17
18
19
20
21
22
58.3
17.6
19.6
45.7
171.1
32.4
2.96 overlapped
1.09 s
58.2
17.6
19.4
45.7
177.7
32.4
2.95 overlapped
1.14 s
0.92 overlapped
2.97 m
0.95 s
2.98 m
2.12 m
3.00 m
2.14 m
23
24
27.3
2.40 m
27.3
2.41 m
2.34 m
2.36 m
125.4
5.35 t (7.2)
125.3
5.38 t (6.5)
29
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