A new glucopyranoside from the leaves of Microsorium fortunei

Article abstract of DOI:10.1080/14786419.2020.1731745

A new compound, 2-hydroxy-4-[3’,5’-dihydroxyhexyl]phenyl-β-D-glucopyranoside (1), together with five known compounds (2–6), were isolated from the leaves of Microsorium fortunei. Their structures were determined by spectroscopic techniques, especially 2D NMR and MS data analyses. All of these compounds are phenolic glycosides and were isolated from this plant for the first time. In addition, compound 1 showed moderate inhibitory activity against α-glucosidase with IC₅₀ value at 0.111 ± 0.061 mg/mL.

Full text of DOI:10.1080/14786419.2020.1731745

Natural Product Research
Formerly Natural Product Letters
ISSN: 1478-6419 (Print) 1478-6427 (Online) Journal homepage: https://www.tandfonline.com/loi/gnpl20
A new glucopyranoside from the leaves of
Microsorium fortunei
Mengqi Yan, Qin Luo, Xianli Zhou, Qinghu Mo, Caifeng Peng, Xiaoya Xian,
Xiao Huang, Xinping Yang, Xu Chen & Chengqin Liang
To cite this article: Mengqi Yan, Qin Luo, Xianli Zhou, Qinghu Mo, Caifeng Peng, Xiaoya Xian,
Xiao Huang, Xinping Yang, Xu Chen & Chengqin Liang (2020): A new glucopyranoside from the
leaves of Microsoriumꢀfortunei, Natural Product Research, DOI: 10.1080/14786419.2020.1731745
To link to this article: https://doi.org/10.1080/14786419.2020.1731745
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NATURAL PRODUCT RESEARCH
https://doi.org/10.1080/14786419.2020.1731745
A new glucopyranoside from the leaves of
Microsorium fortunei
a
ꢀ
c
ꢀ
b
a
a
Mengqi Yan , Qin Luo , Xianli Zhou , Qinghu Mo , Caifeng Peng , Xiaoya
a
a
a
a
a
Xian , Xiao Huang , Xinping Yang , Xu Chen and Chengqin Liang
a
b
College of Pharmacy, Guilin Medical University, Guilin, P. R. China; College of Biotechnology,
c
Guilin Medical University, Guilin, P. R. China; Science Experiment Center, Guilin Medical University,
Guilin, P. R. China
ABSTRACT
ARTICLE HISTORY
A new compound, 2-hydroxy-4-[3’,5’-dihydroxyhexyl]phenyl-b-D-
glucopyranoside (1), together with five known compounds (2–6),
were isolated from the leaves of Microsorium fortunei. Their struc-
tures were determined by spectroscopic techniques, especially 2D
NMR and MS data analyses. All of these compounds are phenolic
glycosides and were isolated from this plant for the first time.
In addition, compound 1 showed moderate inhibitory activity
against a-glucosidase with IC50 value at 0.111 ± 0.061 mg/mL.
Received 8 December 2019
Accepted 26 January 2020
KEYWORDS
Microsorium fortunei;
glucopyranoside; phenolic
glycoside; a-glucosidase
1. Introduction
Microsorium fortunei (T. Moore) Ching (¼M. fortunei), belonging to the family
Polypodiaceae, grows in south of the Yangtze river (Sun and Cheng. 2004). The whole
plant can be used to treat pain of body surface or internal organs and snake bite,
scorpion venom, burns, etc. (Yao et al. 2005). It is also used for the treatment of stom-
ach cold pain, bronchial asthma, rheumatic pain, gonococcal disease and dysentery by
some ethnic minorities, such as Tujia, Miao, Mulao and Lisu nationality (Wei et al.
1999). Several biological activities have been reported for extracts of this plant such as
cure jaundice, urinary tract infection and bruises (Wu et al. 2015). Some flavonoids
and phenolic acids were found from this species in our previous phytochemical
CONTACT Xianli Zhou
These authors contribute to the paper equally.
xlzhou2009@163.com; Chengqin Liang
cqliang@glmc.edu.cn
ꢀ
Supplemental data for this article can be accessed at https://doi.org/10.1080/14786419.2020.1731745.
ß 2020 Informa UK Limited, trading as Taylor & Francis Group

2
M. YAN ET AL.
studies (Liang et al. 2017). Motivated by a search for bioactive metabolites from this
plant, a reinvestigation of the chemical constituents was carried out. As a result, an
unprecedented glucopyranoside (1) was isolated from this plant. In this paper, we report
the isolation, structure elucidation, and biological activities of the new compound.
2. Results and discussion
The 75% ethanol extract of the leaves of M. fortunei was partitioned with EtOAc and
n-BuOH, successively. The n-BuOH-soluble portion was separated by a combination of
silica gel, repeated medium-pressure liquid chromatography and HPLC to afford a new
glucopyranoside (1) and five known compounds (2–6) (Figure 1). The known com-
pounds, compared with literature, were identified as 4-O-b-D-glucopyranosyl- caffeic
acid (2) (Cui et al. 1990), baihuaqianhuoside (3) (Kong et al. 1993), isovitexin (4) (Calis
0
0
0
0
et al. 2007), lispedin (5) (F ꢀa tima et al. 2003), 4,4 -dimethoxy-3 -hydroxy-7, 9 ,7 ,9-die-
poxylignan-3-O-b-D-glucopyranoside (6) (Li et al. 2003).
Compound 1 was obtained as a yellow amorphous powder with [a]20 D ꢁ 37.3 (c
1
4
.0, MeOH). Its molecular formula was established as C H O by HRESIMS at m/z
18 28 9
þ
11.1831 [M þ Na] (calcd for 411.1631), indicating five degrees of unsaturation. The IR
ꢁ
1
spectrum showed absorption bands of hydroxy (3368 cm ) and benzene group (1508
ꢁ
1
1
and 1596 cm ). The H-NMR spectrum (Figure S4) showed the presence of three aro-
matic protons at d 6.61 (dd, J ¼ 8.2, 2.1 Hz, 1 H), 6.69 (d, J ¼ 2.1 Hz, 1 H) and 7.06 (d,
H
J ¼ 8.2 Hz, 1 H), one methyl at d 1.15 (d, J ¼ 6.3 Hz, 3 H), and an anomeric proton at d
H
H
13
4
.68 (d, J ¼ 7.2 Hz, 1 H). The C NMR and DEPT spectra (Figure S5) exhibited 18 carbon
resonances, including one methyl carbon (d 24.3), three methylene carbons (d 47.3,
C
C
4
7
1
1.0, 32.4), one oxymethylene carbon (d 62.4), seven oxymethine carbons (d 104.7,
C
C
8.2, 77.6, 74.9, 71.3, 68.7, 65.5), six phenyl carbons (d 148.3, 144.9, 139.6, 120.9, 119.1,
C
17.2). These data indicated there was a benzene ring and a sugar unit.
OH
6
'
5
'
O
OH
4
'
OH
O
O
3
'
2
'
OH
1
'
4
HO
O
O
O
HO
5
6
3
OCH3
2
OH
HO
O
HO
OH
1
O
HO
HO
OH
O
OH
O
6
''
HO
HO
OH
O
''
5
HO4''
1''
OH
HO
2
'' OH
HO
HOHO 3''
1
2
3
4
OH
OH
O
O
O
OCH3
O
HO
HO
O
HO
HO
O
O
H
H
OH
HO
O
OH
HO
OH
H CO
O
O
HO
3
OH
5
6
Figure 1. Structures of compounds 1-6 from M. fortunei.

NATURAL PRODUCT RESEARCH
3
The signals of the three aromatic protons at d 6.61 (1 H, dd, J ¼ 8.2, 2.1 Hz, H-5),
H
6
.69 (1 H, d, J ¼ 2.1 Hz, H-3) and 7.06 (1 H, d, J ¼ 8.2 Hz, H-6) constituteda classical ABX
system, indicated the presence of a 1,3,4-trisubstituted aromatic ring. The aromatic
ring was also established through the HMBC correlations (Figure S1) of H-3 to C-1 (dC
1
44.9), C-2 (d 148.3) and C-5 (d 120.9), H-5 to C-1 (d 144.9) and C-3 (d 117.2) and
C C C C
H-6 to C-1 (d 144.9), C-2 (d 148.3) and C-4 (d 139.6). The sugar unit was confirmed
C
C
C
to be D-glucose based on acid hydrolysis of compound 1 and HPLC analysis of its
derivative (Supporting information). The configuration of the D-glucose was deter-
mined to be b from its anomeric proton coupling constant at d 4.68 (1 H, d,
H
0
0
J ¼ 7.2 Hz, H-1 ’) (Agrawal 1992). And the HMBC correlations of H-1 ’ with C-1 (d 144.9)
C
indicated the sugar unit was located at C-1.
Extensive analysis of the NMR spectroscopic data of 1 showed a close resemblance
with 2-hydroxy-4-[(3S)ꢁ3-hydroxybutyl]phenyl-b-D-glucopyranoside (Shimoda et al. 2007).
The most prominent differences of them were the branched chains attached to the aro-
0
0
0
0
0
0
1
1
matic ring. The correlations of H -1 /H -2 /H-3 /H -4 /H-5 /H -6 in the H- H COSY spec-
2
2
2
3
0
0
trum (Figure S1), indicated that the branched chain of 1 was a 3 ,5 -dioxyhexylmoiety. The
HMBC correlations of H -1 [d 2.64 (m, 1 H) and d 2.52 (m, 1 H)] with C-3 (d 117.2), C-4
d 139.6) and C-5 (d 120.9), indicated the 3 ,5 -dioxyhexyl moiety was located at C-4.
0
2
H
H
C
0
0
(
C
C
Considering the molecular formula of compound 1, in addition to one hydroxyl
group attached to C-2 on the benzene ring, there should be two other hydroxyl
0
0
groups which attached to C-3 and C-5 , respectively. On the basis of the above ana-
0
0
lysis, the structure of compound 1 was determined to be 2-hydroxy-4-[3 ,5 -dihydroxy-
hexyl] phenyl-b-D-glucopyranoside.
0
0
In addition, there is a pair of intersite hydroxyl groups in 3 ,5 -dihydroxyhexyl moiety,
the relative configuration of them still could not be determined by ROESY, Mosher’s,
induced CD, and dimolybdenum tetraacetate methods (Li and Cui. 2015). Thus, the
structure still require solid evidence, such as X-ray analysis, or comparison with synthetic
compounds, for unequivocal assignment. Unfortunately, we could not obtain crystal for
X-ray analysis, and synthesize the corresponding compounds for the comparison.
The a-glucosidase inhibitory effect of compound 1 was evaluated along with the clin-
ical a-glucosidase inhibitor acarbose. The results showed the inhibition effect of compound
1
was found to be dose-dependent like as acarbose (Figure S2a) with IC50 value of
0
.111 ± 0.061 mg/mL (acarbose ¼ 0.235 ± 0.022 mg/mL). According to in Supplementary
information Figure S2b, when the mass concentration of compound 1 was 0.5 mg/mL, the
enzyme activity could be rapidly inhibited within 1 min, and the inhibition rate can reach
a maximum, and then decreased. In addition, in the enzyme kinetics experiment, the
results showed that compound 1 is a reversible inhibitor, because the increment in com-
pound 1 concentration resulted in lowering the slope of the lines (Figure S3a) (Wei et al.
2017). According to Figure S3c, the inhibition of compound 1 on the enzyme showed a
non-competitive inhibition type (Wei et al. 2017) with a K of 6.570 mg/mL.
m
3. Experimental
3.1. General experimental procedures
IR was measured by BRUKER TENSOR27 infrared spectrometer, KBr compression; UV
was determined by Shimadzu UV2401PC UV spectrometer; LC-Quadrupole Mass

4
M. YAN ET AL.
Spectrometer (LC-MS8030, Shimadzu, Japan); HR-ESI-MS spectrum was determined on
Liquid Chromatography Mass Spectrometry (Exavtie, Thermo Fisher Scientific); 1D and
2
D NMR spectra were performed on Bruker DRX-500 MHz and avance III 600 MHz;
CombiFlash Rf (RF200, Teledyne Isco, Inc., USA); Agilent Technologies (Agilent LC1260
infinity, Agilent); Rotary Evaporator (RE-52A, Shanghai Yarong Biochemical Instrument
Factory); Electronic Balance (BS400S, Beijing Sartorius Co., Ltd.; Mettler Toledo XS105
DualRange, USA); Silica gel (100–200 mesh, Qingdao Marine Chemical Inc., China) were
used for column chromatography (CC); TLC was conducted with glass precoated with
silica gel GF254 (Qingdao Marine Chemical Inc., China); Inverting filler (RP-18, Merk).
MCI filler (MCI-gel CHP-20P, Mitsubishi, Japan); RP column (Zorbax SB-C18, 5 lm,
9
.4 ꢂ 250 mm, Agilent, USA); D-glucose and 1-Phenyl-3-methyl-5-pyrazolone (PMP)
(
(
Aladdin Co., Shanghai, China); Chloroform, petroleum ether, ethyl acetate, methanol
AR, all purchased from Xilong Scientific Co., Ltd., China); a-Glycosidase (G0660-750UN,
SIGMA, Germany); Acarbose (109A032, solarbio, Beijing Solarbio Technology Co., Ltd.);
-pNPG (N0493, Tokyo Chemical Industry Co., Ltd.).
4
3.2. Plant material
Dried leaves of M. fortunei were collected from Ziyuan country of Guangxi Zhuang
Autonomous Region, China and identified by professor Yunqiu Li (Guilin Medical
University, College of Pharmacy). The voucher specimen (No. 2016101201) has been
deposited in the Institute of Traditional Chinese Medicine and Natural Products,
College of Pharmacy, Guilin Medical University (Guilin, China).
3
.3. Extraction and isolation
The power-dried leaves of M. fortunei (40 kg) were heated for reflux extractions with
5% ethanol three times, each time for 1 hour. The extract was concentrated in vacuo
7
to afford a brown reside. Then it was suspended in H O, and partitiomed with EtOAc
2
and n-BuOH, succsssively. The n-BuOH extraction was applied to silica gel (100-200
mesh) column chromatography, eluting with a CHCl -CH OH gradient system (v/v, 1:0,
3
3
9
:1, 8:2, 2:1, 1:1, 0:1), to yiled six fractions (Fr.A ꢃ Fr.F) based on the TLC analysis. The
fraction B was subjected by CombiFlash Rf 200 (flow rate 10.0 mL/min), eluted with
CH OH-H O gradient system 10% ꢃ 50%, yielded fraction B1 ꢃ B5. The further separ-
3
2
ation of Fr.B3 was separated by Agilent LC1260 HPLC (flow rate 1.0 mL/min, 17%
CH OH-H O) to afford compound 3 (16.8 mg). The Fr.B5 was subjected by Agilent
3
2
LC1260 HPLC (flow rate 1.0 mL/min, 13% CH CN-H O) to afford compound 6 (18.2 mg).
3
2
The fraction C was subjected by silica gel conlumn chromatography, eluted with
CHCl -CH OH gradient system (v/v, 5:1), yielded forty major fractions (Fr.C1 ꢃ Fr.C40).
3
3
The further separation of Fr.C13 was subjected by Agilent LC1260 (flow rate 1.0 mL/
min, 25% CH OH-H O) to afford compound 4 (16.1 mg). And after merged together by
3
2
analyzing TLC characteristics, the Fr.C30 ꢃ Fr.C34 were subjected by Agilent LC1260
HPLC (flow rate 1.0 mL/min, 20% CH CN-H O) to afford compound 1 (20.8 mg) and
3
2
2
(16.6 mg).

NATURAL PRODUCT RESEARCH
5
3.3.1. 2-hydroxy-4-[3’,5’-dihydroxyhexyl]phenyl-b-D-glucopyranoside
Obtained as yellow powder with [a]20 D ꢁ 37.3 (c 1.0, MeOH); UV (MeOH), k
(log e)
max
2
03 (4.47) nm; IR (KBr) V
3368, 2925, 1596, 1508, 1384, 1277, 1072, 868, 803,
max
ꢁ
1
1
6
17 cm
;
H-NMR (500 MHz, MeOD) d : 7.06 (1 H, d, J ¼ 8.2 Hz, H-6), 6.69 (1 H, d,
H
0
J ¼ 2.1 Hz, H-3), 6.61 (1 H, dd, J ¼ 8.2, 2.1 Hz, H-5), 4.68 (1 H, d, J ¼ 7.2 Hz, H-1 ’), 3.96 (1 H,
0
0
0
m, H-5 ), 3.86 (1 H, dd, J ¼ 12.1, 1.7 Hz, H-6 ’a), 3.76 (1 H, m, H-3 ), 3.70 (1 H, dd, J ¼ 12.1,
0 0 0 0 0
4
.5 Hz, H-6 ’b), 3.47 (1 H, m, H-3 ’), 3.44 (1 H, m, H-2 ’), 3.37 (2 H, m, H-4 ’, H-5 ’), 2.64 (1 H,
0
0
m, H-1’a), 2.52 (1 H, m, H-1’b), 1.66 (2 H, m, H-2 ), 1.49 (2 H, m, H-4 ), 1.15 (3 H, d,
0
13
J ¼ 6.3 Hz, H-6 )。 C-NMR (125 MHz, MeOD) d : 144.9 (C-1), 148.3 (C-2), 117.2 (C-3), 139.6
C
0 0 0 0 0
(
2
C-4), 120.9 (C-5), 119.1 (C-6), 32.4 (C-1 ), 41.0 (C-2 ), 68.7 (C-3 ), 47.3 (C-4 ), 65.5 (C-5 ),
0 0 0 0 0 0 0
4.3 (C-6 ), 104.7 (C-1 ’), 77.6 (C-2 ’), 74.9 (C-3 ’), 71.3 (C-4 ’), 78.2 (C-5 ’), 62.4 (C-6 ’). ESI-MS
þ
þ
m/z 411 [M þ Na] ; HR-ESI-MS m/z 411.1831 [M þ Na] (calcd for C H O Na, 411.1631).
18 28 9
3.4. Acid hydrolysis and HPLC analysis of compound 1
The absolute configuration of the sugar unit was determined by the method of pre-col-
umn derivatization with PMP-High Performance Liquid Chromatography (HPLC) with
slight modifications (Li et al. 2013). Compound 1 afforded D-glucose (t ¼ 34.25 min).
R
3.5. Bioactivity assay
The assay was performed as reported method (Didem et al. 2017) with the positive
control of acarbose. Briefly, a mixture of 50 lL of different concentrations (0.001, 0.01,
0
6
.05, 0.1, 0.2, 0.5 mg/mL) of the test samples and 100 lL of 0.1 M phosphate buffer (pH
.9) containing yeast a-glucosidase solution (0.5 U/mL) was incubated in 96-well plates
ꢄ
at 25 C for 15 min, then 50 mL of 5 mM 4-pNPG was added into the mixture and inter-
act at 25 C for another 10 min. The absorbance was recorded at 405 nm (1, 5, 10, 15,
ꢄ
20 min) by a microplatereader (Infinite M200 Pro, Tecan Corp., Switzerland). a-glucosi-
dase inhibition (%) ¼ [(A - Asample)/Acontrol] ꢂ 100%. Where A is the activity
control
control
of enzyme without compound/positive and A
is the activity of enzyme with com-
sample
pound/positive at different concentrations. The inhibitory mechanism assay was
applied with varying the concentration of the enzyme in the reaction mixture, and the
inhibition type was then assayed by the Lineweaver–Burk plot.
4. Conclusion
In summary, comparing with the positive control acarbose, we found that the new
0
0
compound of 2-hydroxy-4-[3 ,5 -dihydroxyhexyl]phenyl-b-D-glucopyranoside isolated
from the leaves of M. fortunei possessed potent a-glucosidase inhibitory activity with
an IC50 value of 0.111 ± 0.061 mg/mL. This compound reversibly inhibited the enzyme
in a non-competitive manner. Hence, the new compound identified in this work may
promise candidates for developing as novel anti-diabetic agents.
Acknowledgement
We are grateful to Guangxi Normal University, for measuring the IR, UV, MS and NMR spectra.

6
M. YAN ET AL.
Disclosure statement
No potential conflict of interest was reported by the authors.
Funding
This work was supported by the [National Natural Science Foundation of China] under Grant
[No. 31560100; No. 81860621]; [Natural Science Fund of Guangxi Province] under Grant [No.
2
2
017GXNSFAA198242;
No.
2018GXNSFBA281032;
No.
2018GXNSFAA281102;
No.
018GXNSFBA281079], and [State Key Laboratory for Chemistry and Molecular Engineering of
Medicinal Resources] (Guangxi Normal University) under Grant [No. CHEMR2017-B15].
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